Therapeutic DLL4 Binding Proteins

ABSTRACT

DLL4 binding proteins are described herein, including antibodies, CDR-grafted antibodies, humanized antibodies, and DLL4 binding fragments thereof, proteins that bind DLL4 with high affinity, and DLL4 binding proteins that neutralize DLL4 and/or VEGF activity. The DLL4 binding proteins are useful for treating or preventing cancers and tumors and especially for treating or preventing tumor angiogenesis.

REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of Ser. No. 15/582,112 which is aContinuation U.S. patent application Ser. No. 15/295,427 filed Oct. 17,2016, which is a Continuation of U.S. patent application Ser. No.14/798,849, filed Jul. 14, 2015, now U.S. Pat. No. 9,469,689, issuedOct. 18, 2016, which is a Division of U.S. patent application Ser. No.13/037,932, filed Mar. 1, 2011, now U.S. Pat. No. 9,115,195, issued Aug.25, 2015, which claims the benefit of priority to U.S. ProvisionalApplication No. 61/309,494, filed Mar. 2, 2010. The contents of each ofthe above priority documents are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the development and use of DLL4 bindingproteins and uses thereof in the inhibition, prevention, and/ortreatment of cancers, tumors, other angiogenesis-dependent diseases,angiogenesis-independent diseases, and macular degeneration andage-related macular degeneration diseases characterized by aberrant DLL4expression or activity.

BACKGROUND OF THE INVENTION

Cell-to-cell communication is required for many biological processessuch as differentiation, proliferation, and homeostasis. One systemutilized by a wide range of eukaryotes is the Notch-signaling pathway.This pathway, especially the Notch receptor, is also critical forfunctional tumor angiogenesis. Thus, inhibition of Notch receptorfunction, blockage of the Notch receptor, and/or blockage of theNotch-signaling pathway are potential strategies for anti-cancercompositions and therapies. Small molecule inhibitors of the Notchreceptor have proven to be toxic because they suppress wild type(normal) tissue expression of Notch receptors throughout the body. Thus,different members of the Notch-signaling pathway should be considered aspotential targets for therapeutics.

A vasculature ligand for the Notch receptor is Delta 4 or Delta-like 4(DLL4). Largely expressed in the vasculature, DLL4 is critical forvascular development (Yan et al., Clin. Cancer Res., 13(24): 7243-7246(2007); Shutter et al., Genes Dev., 14(11): 1313-1318 (2000); Gale etal., Proc. Natl. Acad. Sci. USA, 101(45): 15949-15954 (2004); Krebs etal., Genes Dev., 14(11): 1343-1352 (2000)). Mice heterozygous for DLL4are embryonically lethal due to major defects in vascular development(Gale et al., Proc. Natl. Acad. Sci. USA, 101(45): 15949-15954 (2004);Duarte et al., Genes Dev., 18(20): 2474-2478 (2004); Krebs et al., GenesDev., 18(20): 2469-2473 (2004)). The expression of DLL4 can be inducedby VEGF (Liu et al., Mol. Cell Biol., 23(1): 14-25 (2003); Lobov et al.,Proc. Natl. Acad. Sci. USA, 104(9): 3219-3224 (2007)). In sum, DLL4 cannegatively regulate VEGF signaling, in part through repressing VEGFR2and inducing VEGFR1 (Harrington et al., Microvasc. Res., 75(2): 144-154(2008); Suchting et al., Proc. Natl. Acad. Sci. USA, 104(9): 3225-3230(2007)). Exquisite coordination between DLL4 and VEGF is essential forfunctional angiogenesis.

In addition to its physiological role, DLL4 is up-regulated in tumorblood vessels (Gale et al., Proc. Natl. Acad. Sci. USA, 101(45):15949-15954 (2004); Mailhos et al., Differentiation, 69(2-3): 135-144(2001); Patel et al., Cancer Res., 65(19): 8690-8697 (2005); Patel etal., Clin. Cancer Res., 12(16): 4836-4844 (2006); Noguera-Troise et al.,Nature, 444(7122): 1032-1037 (2006)). Blockade of DLL4 potentlyinhibited primary tumor growth in multiple models (Noguera-Troise etal., Nature, 444(7122): 1032-1037 (2006); Ridgway et al., Nature,444(7122): 1083-1087 (2006); Scehnet et al., Blood, 109(11): 4753-4760(2007)). The inhibition of DLL4 was even effective against tumors thatare resistant to anti-VEGF therapy. The combinatorial inhibition of bothDLL4 and VEGF provided an enhanced anti-tumor activity. Interestingly,unlike VEGF inhibition that reduces tumor vessel formation, DLL4blockade leads to an increase in tumor vasculature density wherein thevessels are abnormal, cannot support efficient blood transport, and areeffectively nonfunctional. Thus, DLL4 provides a potential target forcancer treatment.

There is a need in the art for therapeutic agents capable of targetingthe DLL4-Notch pathway and thereby inhibiting, or even preventing, tumorangiogenesis and growth.

SUMMARY OF THE INVENTION

The invention provides proteins that bind human DLL4. DLL4 bindingproteins of the invention include, but are not limited to, ratmonoclonal antibodies, chimeric antibodies, CDR-grafted antibodies,humanized antibodies, primate-ized antibodies, affinity maturedantibodies, and fragments thereof that are capable of binding humanDLL4. Preferably, a binding protein described herein binds human DLL4with high affinity. More preferably, a binding protein according to theinvention is capable of neutralizing human DLL4. The invention alsoprovides methods of making and using DLL4 binding proteins.

One aspect of the invention provides a binding protein capable ofbinding human DLL4, wherein the binding protein comprises at least oneamino acid sequence selected from the group of amino acid sequencesconsisting of SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ IDNO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, and SEQ ID NO:164.

An aspect of this invention pertains to a binding protein comprising anantigen binding domain wherein the binding protein is capable of bindinghuman DLL4, said antigen binding domain comprising at least one or more(i.e., two, three, four, five, or six) CDRs wherein:

CDR-H1 is selected from the group consisting of:

-   -   X₁—X₂—X₃—X₄—X₅ (SEQ ID NO:151), wherein;        -   X₁ is N, H, or Y;        -   X₂ is F;        -   X₃ is P;        -   X₄ is M;        -   X₅ is A or S;    -   residues 31-35 of SEQ ID NO:157 (CDR-H1 38H12);    -   residues 31-35 of SEQ ID NO:161 (CDR-H1 37D10);    -   residues 31-35 of SEQ ID NO:163 (CDR-H1 32C7);    -   residues 31-35 of SEQ ID NO:165 (CDR-H1 14G1);    -   residues 31-35 of SEQ ID NO:167 (CDR-H1 14A11);    -   residues 31-35 of SEQ ID NO:169 (CDR-H1 15D6);    -   residues 31-35 of SEQ ID NO:171 (CDR-H1 VH.1 1A11);    -   residues 31-35 of SEQ ID NO:172 (CDR-H1 VH.1a 1A11);    -   residues 31-35 of SEQ ID NO:173 (CDR-H1 VH.1b 1A11);    -   residues 31-35 of SEQ ID NO:174 (CDR-H1 VH.2a 1A11);    -   residues 31-35 of SEQ ID NO:179 (CDR-H1 VH.1 38H12);    -   residues 31-35 of SEQ ID NO:180 (CDR-H1 VH.1A 38H12);    -   residues 31-35 of SEQ ID NO:181 (CDR-H1 VH.1b 38H12);    -   residues 31-35 of SEQ ID NO:182 (CDR-H1 VH.2a 38H12);    -   residues 31-35 of SEQ ID NO:187 (CDR-H1 h1A11VH.1);    -   residues 31-35 of SEQ ID NO:188 (CDR-H1 h1A11.A6);    -   residues 31-35 of SEQ ID NO:189 (CDR-H1 h1A11.A8);    -   residues 31-35 of SEQ ID NO:190 (CDR-H1 h1A11.C6);    -   residues 31-35 of SEQ ID NO:191 (CDR-H1 h1A11.A11);    -   residues 31-35 of SEQ ID NO:192 (CDR-H1 h1A11.B5);    -   residues 31-35 of SEQ ID NO:193 (CDR-H1 h1A11.E12);    -   residues 31-35 of SEQ ID NO:194 (CDR-H1 h1A11.G3);    -   residues 31-35 of SEQ ID NO:195 (CDR-H1 h1A11.F5); and    -   residues 31-35 of SEQ ID NO:196 (CDR-H1 h1A11.H2);

CDR-H2 is selected from the group consisting of:

-   -   X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—X₉—X₁₀—X₁₁—X₁₂—X₁₃—X₁₄—X₁₅—X₁₆—X₁₇ (SEQ        ID NO:152), wherein;        -   X₁ is T or S;        -   X₂ is I;        -   X₃ is S;        -   X₄ is S or G;        -   X₅ is S;        -   X₆ is D;        -   X₇ is G, A, D, S, or E;        -   X₈ is T or W;        -   X₉ is T, P, or A;        -   X₁₀ is Y, S, T, or N;        -   X₁₁ is Y or I;        -   X₁₂ is R or G;        -   X₁₃ is D;        -   X₁₄ is S;        -   X₁₅ is V;        -   X₁₆ is K; and        -   X₁₇ is G;    -   residues 50-66 of SEQ ID NO:157 (CDR-H2 38H12);    -   residues 50-68 of SEQ ID NO:161 (CDR-H2 37D10);    -   residues 50-66 of SEQ ID NO:163 (CDR-H2 32C7);    -   residues 50-66 of SEQ ID NO:165 (CDR-H2 14G1);    -   residues 50-66 of SEQ ID NO:167 (CDR-H2 14A11);    -   residues 50-66 of SEQ ID NO:169 (CDR-H2 15D6);    -   residues 50-66 of SEQ ID NO:171 (CDR-H2 VH.1 1A11);    -   residues 50-66 of SEQ ID NO:172 (CDR-H2 VH.1a 1A11);    -   residues 50-66 of SEQ ID NO:173 (CDR-H2 VH.1b 1A11);    -   residues 50-66 of SEQ ID NO:174 (CDR-H2 VH.2a 1A11);    -   residues 50-66 of SEQ ID NO:179 (CDR-H2 VH.1 38H12);    -   residues 50-66 of SEQ ID NO:180 (CDR-H2 VH.1A 38H12);    -   residues 50-66 of SEQ ID NO:181 (CDR-H2 VH.1b 38H12);    -   residues 31-35 of SEQ ID NO:182 (CDR-H1 VH.2a 38H12);    -   residues 50-66 of SEQ ID NO:187 (CDR-H2 h1A11VH.1);    -   residues 50-66 of SEQ ID NO:188 (CDR-H2 h1A11.A6);    -   residues 50-66 of SEQ ID NO:189 (CDR-H2 h1A11.A8);    -   residues 50-66 of SEQ ID NO:190 (CDR-H2 h1A11.C6);    -   residues 50-66 of SEQ ID NO:191 (CDR-H2 h1A11.A11);    -   residues 50-66 of SEQ ID NO:192 (CDR-H2 h1A11.B5);    -   residues 50-66 of SEQ ID NO:193 (CDR-H2 h1A11.E12);    -   residues 50-66 of SEQ ID NO:194 (CDR-H2 h1A11.G3);    -   residues 50-66 of SEQ ID NO:195 (CDR-H2 h1A11.F5); and    -   residues 50-66 of SEQ ID NO:196 (CDR-H2 h1A11.H2);

CDR-H3 is selected from the group consisting of:

-   -   X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—X₉ (SEQ ID NO:153), wherein;        -   X₁ is G;        -   X₂ is Y;        -   X₃ is Y;        -   X₄ is N;        -   X₅ is S;        -   X₆ is P;        -   X₇ is F;        -   X₈ is A; and        -   X₉ is Y, F, or S;    -   residues 99-107 of SEQ ID NO:157 (CDR-H3 38H12);    -   residues 101-111 of SEQ ID NO:161 (CDR-H3 37D10);    -   residues 99-105 of SEQ ID NO:163 (CDR-H3 32C7);    -   residues 99-105 of SEQ ID NO:165 (CDR-H3 14G1);    -   residues 99-110 of SEQ ID NO:167 (CDR-H3 14A11);    -   residues 99-110 of SEQ ID NO:169 (CDR-H3 15D6);    -   residues 99-107 of SEQ ID NO:171 (CDR-H3 VH.1 1A11);    -   residues 99-107 of SEQ ID NO:172 (CDR-H3 VH.1a 1A11);    -   residues 99-107 of SEQ ID NO:173 (CDR-H3 VH.1b 1A11);    -   residues 99-107 of SEQ ID NO:174 (CDR-H3 VH.2a 1A11);    -   residues 99-107 of SEQ ID NO:179 (CDR-H3 VH.1 38H12);    -   residues 99-107 of SEQ ID NO:180 (CDR-H3 VH.1A 38H12);    -   residues 99-107 of SEQ ID NO:181 (CDR-H2 VH.1b 38H12);    -   residues 99-107 of SEQ ID NO:182 (CDR-H1 VH.2a 38H12);    -   residues 99-107 of SEQ ID NO:187 (CDR-H3 h1A11VH.1);    -   residues 99-107 of SEQ ID NO:188 (CDR-H3 h1A11.A6);    -   residues 99-107 of SEQ ID NO:189 (CDR-H3 h1A11.A8);    -   residues 99-107 of SEQ ID NO:190 (CDR-H3 h1A11.C6);    -   residues 99-107 of SEQ ID NO:191 (CDR-H3 h1A11.A11);    -   residues 99-107 of SEQ ID NO:192 (CDR-H3 h1A11.B5);    -   residues 99-107 of SEQ ID NO:193 (CDR-H3 h1A11.E12);    -   residues 99-107 of SEQ ID NO:194 (CDR-H3 h1A11.G3);    -   residues 99-107 of SEQ ID NO:195 (CDR-H3 h1A11.F5); and    -   residues 99-107 of SEQ ID NO:196 (CDR-H3 h1A11.H2);

CDR-L1 is selected from the group consisting of:

-   -   X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—X₉—X₁₀—X₁₁ (SEQ ID NO:154), wherein;        -   X₁ is R;        -   X₂ is A;        -   X₃ is S;        -   X₄ is E or Q;        -   X₅ is D or E;        -   X₆ is I;        -   X₇ is Y or W;        -   X₈ is S, I, Y, N, or R;        -   X₉ is N;        -   X₁₀ is L; and        -   X₁₁ is A;    -   residues 24-34 of SEQ ID NO:158 (CDR-L1 38H12);    -   residues 24-34 of SEQ ID NO:162 (CDR-L1 37D10);    -   residues 24-34 of SEQ ID NO:164 (CDR-L1 32C7);    -   residues 24-34 of SEQ ID NO:166 (CDR-L1 14G1);    -   residues 23-37 of SEQ ID NO:168 (CDR-L1 14A11);    -   residues 23-37 of SEQ ID NO:170 (CDR-L1 15D6);    -   residues 24-34 of SEQ ID NO:175 (CDR-L1 VL.1 1A11);    -   residues 24-34 of SEQ ID NO:176 (CDR-L1 VL.1a 1A11);    -   residues 24-34 of SEQ ID NO:177 (CDR-L1 VL.1b 1A11);    -   residues 24-34 of SEQ ID NO:178 (CDR-L1 VL.2a 1A11);    -   residues 24-34 of SEQ ID NO:183 (CDR-L1 VL.1 38H12);    -   residues 24-34 of SEQ ID NO:184 (CDR-L1 VL.1a 38H12);    -   residues 24-34 of SEQ ID NO:185 (CDR-L1 VL.1b 38H12);    -   residues 24-34 of SEQ ID NO:186 (CDR-L1 VL.2a 38H12);    -   residues 24-34 of SEQ ID NO:197 (CDR-L1 h1A11VL.1);    -   residues 24-34 of SEQ ID NO:198 (CDR-L1 h1A11.A2);    -   residues 24-34 of SEQ ID NO:199 (CDR-L1 h1A11.A12);    -   residues 24-34 of SEQ ID NO:200 (CDR-L1 h1A11.A7);    -   residues 24-34 of SEQ ID NO:201 (CDR-L1 h1A11.B4);    -   residues 24-34 of SEQ ID NO:202 (CDR-L1 h1A11.B5); and    -   residues 24-34 of SEQ ID NO:203 (CDR-L1 h1A11.E12);

CDR-L2 is selected from group consisting of:

-   -   X₁—X₂—X₃—X₄—X₅—X₆—X₇ (SEQ ID NO:155), wherein;        -   X₁ is D;        -   X₂ is T;        -   X₃ is N or S;        -   X₄ is N, D, S, I, Y, or V;        -   X₅ is L;        -   X₆ is A; and        -   X₇ is D;    -   residues 50-56 of SEQ ID NO:158 (CDR-L2 38H12);    -   residues 50-56 of SEQ ID NO:162 (CDR-L2 37D10);    -   residues 50-56 of SEQ ID NO:164 (CDR-L2 32C7);    -   residues 50-56 of SEQ ID NO:166 (CDR-L2 14G1);    -   residues 53-59 of SEQ ID NO:168 (CDR-L2 14A11);    -   residues 53-59 of SEQ ID NO:170 (CDR-L2 15D6);    -   residues 50-56 of SEQ ID NO:175 (CDR-L2 VL.1 1A11);    -   residues 50-56 of SEQ ID NO:176 (CDR-L2 VL.1a 1A11);    -   residues 50-56 of SEQ ID NO:177 (CDR-L2 VL.1b 1A11);    -   residues 50-56 of SEQ ID NO:178 (CDR-L2 VL.2a 1A11);    -   residues 50-56 of SEQ ID NO:183 (CDR-L2 VL.1 38H12);    -   residues 50-56 of SEQ ID NO:184 (CDR-L2 VL.1a 38H12);    -   residues 50-56 of SEQ ID NO:185 (CDR-L2 VL.1b 38H12);    -   residues 50-56 of SEQ ID NO:186 (CDR-L2 VL.2a 38H12);    -   residues 50-56 of SEQ ID NO:197 (CDR-L2 h1A11VL.1);    -   residues 50-56 of SEQ ID NO:198 (CDR-L2 h1A11.A2);    -   residues 50-56 of SEQ ID NO:199 (CDR-L2 h1A11.A12);    -   residues 50-56 of SEQ ID NO:200 (CDR-L2 h1A11.A7);    -   residues 50-56 of SEQ ID NO:201 (CDR-L2 h1A11.B4);    -   residues 50-56 of SEQ ID NO:202 (CDR-L2 h1A11.B5); and    -   residues 50-56 of SEQ ID NO:203 (CDR-L2 h1A11.E12); and

CDR-L3 is selected from the group consisting of:

-   -   X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—X₉ (SEQ ID NO:156), wherein;        -   X₁ is Q;        -   X₂ is Q;        -   X₃ is Y;        -   X₄ is N, D, or T;        -   X₅ is N, Y, or W;        -   X₆ is Y or V;        -   X₇ is P;        -   X₈ is P; and        -   X₉ is T.    -   residues 89-97 of SEQ ID NO:158 (CDR-L3 38H12);    -   residues 89-97 of SEQ ID NO:162 (CDR-L3 37D10);    -   residues 89-97 of SEQ ID NO:164 (CDR-L3 32C7);    -   residues 89-98 of SEQ ID NO:166 (CDR-L3 14G1);    -   residues 92-100 of SEQ ID NO:168 (CDR-L3 14A11);    -   residues 92-100 of SEQ ID NO:170 (CDR-L3 15D6);    -   residues 89-97 of SEQ ID NO:175 (CDR-L3 VL.1 1A11);    -   residues 89-97 of SEQ ID NO:176 (CDR-L3 VL.1a 1A11);    -   residues 89-97 of SEQ ID NO:177 (CDR-L3 VL.1b 1A11);    -   residues 89-97 of SEQ ID NO:178 (CDR-L3 VL.2a 1A11);    -   residues 89-97 of SEQ ID NO:183 (CDR-L3 VL.1 38H12);    -   residues 89-97 of SEQ ID NO:184 (CDR-L3 VL.1a 38H12);    -   residues 89-97 of SEQ ID NO:185 (CDR-L3 VL.1b 38H12);    -   residues 89-97 of SEQ ID NO:186 (CDR-L3 VL.2a 38H12);    -   residues 89-97 of SEQ ID NO:197 (CDR-L3 h1A11VL.1);    -   residues 89-97 of SEQ ID NO:198 (CDR-L3 h1A11.A2);    -   residues 89-97 of SEQ ID NO:199 (CDR-L3 h1A11.A12);    -   residues 89-97 of SEQ ID NO:200 (CDR-L3 h1A11.A7);    -   residues 89-97 of SEQ ID NO:201 (CDR-L3 h1A11.B4);    -   residues 89-97 of SEQ ID NO:202 (CDR-L3 h1A11.B5);    -   residues 89-97 of SEQ ID NO:203 (CDR-L3 h1A11.E12).    -   Preferably, a DLL4 binding protein of the invention comprises at        least one CDR comprising an amino acid sequence selected from        the group consisting of:    -   residues 31-35 of SEQ ID NO:157 (CDR-H1 38H12); residues 50-66        of SEQ ID NO:157 (CDR-H2 38H12); residues 99-107 of SEQ ID        NO:157 (CDR-H3 38H12);    -   residues 24-34 of SEQ ID NO:158 (CDR-L1 38H12); residues 50-56        of SEQ ID NO:158 (CDR-L2 38H12); residues 89-97 of SEQ ID NO:158        (CDR-L3 38H12);    -   residues 31-35 of SEQ ID NO:159 (CDR-H1 1A11); residues 50-66 of        SEQ ID NO:159 (CDR-H2 1A11); residues 99-107 of SEQ ID NO:159        (CDR-H3 1A11);    -   residues 24-34 of SEQ ID NO:160 (CDR-L1 1A11); residues 50-56 of        SEQ ID NO:160 (CDR-L2 1A11); residues 89-97 of SEQ ID NO:160        (CDR-L3 1A11);    -   residues 31-35 of SEQ ID NO:161 (CDR-H1 37D10); residues 50-68        of SEQ ID NO:161 (CDR-H2 37D10); residues 101-111 of SEQ ID        NO:161 (CDR-H3 37D10);    -   residues 24-34 of SEQ ID NO:162 (CDR-L1 37D10); residues 50-56        of SEQ ID NO:162 (CDR-L2 37D10); residues 89-97 of SEQ ID NO:162        (CDR-L3 37D10);    -   residues 31-35 of SEQ ID NO:163 (CDR-H1 32C7); residues 50-66 of        SEQ ID NO:163 (CDR-H2 32C7); residues 99-105 of SEQ ID NO:163        (CDR-H3 32C7);    -   residues 24-34 of SEQ ID NO:164 (CDR-L1 32C7); residues 50-56 of        SEQ ID NO:164 (CDR-L2 32C7); residues 89-98 of SEQ ID NO:164        (CDR-L3 32C7);    -   residues 31-35 of SEQ ID NO:165 (CDR-H1 14G1); residues 50-66 of        SEQ ID NO:165 (CDR-H2 14G1); residues 99-105 of SEQ ID NO:165        (CDR-H3 14G1);    -   residues 24-34 of SEQ ID NO:166 (CDR-L1 14G1); residues 50-56 of        SEQ ID NO:166 (CDR-L2 14G1); residues 89-98 of SEQ ID NO:166        (CDR-L3 14G1);    -   residues 31-35 of SEQ ID NO:167 (CDR-H1 14A11); residues 50-66        of SEQ ID NO:167 (CDR-H2 14A11); residues 99-110 of SEQ ID        NO:167 (CDR-H3 14A11);    -   residues 23-37 of SEQ ID NO:168 (CDR-L1 14A11); residues 53-59        of SEQ ID NO:168 (CDR-L2 14A11); residues 92-100 of SEQ ID        NO:168 (CDR-L3 14A11);    -   residues 31-35 of SEQ ID NO:169 (CDR-H1 15D6); residues 50-66 of        SEQ ID NO:169 (CDR-H2 15D6); residues 99-110 of SEQ ID NO:169        (CDR-H3 15D6);    -   residues 23-37 of SEQ ID NO:170 (CDR-L1 15D6); residues 53-59 of        SEQ ID NO:170 (CDR-L2 15D6); residues 92-100 of SEQ ID NO:170        (CDR-L3 15D6);    -   residues 31-35 of SEQ ID NO:171 (CDR-H1 VH.1 1A11); residues        50-66 of SEQ ID NO:171 (CDR-H2 VH.1 1A11); residues 99-107 of        SEQ ID NO:171 (CDR-H3 VH.1 1A11); residues 31-35 of SEQ ID        NO:172 (CDR-H1 VH.1a 1A11); residues 50-66 of SEQ ID NO:172        (CDR-H2 VH.1a 1A11); residues 99-107 of SEQ ID NO:172 (CDR-H3        VH.1a 1A11); residues 31-35 of SEQ ID NO:173 (CDR-H1 VH.1b        1A11); residues 50-66 of SEQ ID NO:173 (CDR-H2 VH.1b 1A11);    -   residues 99-107 of SEQ ID NO:173 (CDR-H3 VH.1b 1A11); residues        31-35 of SEQ ID NO:174 (CDR-H1 VH.2a 1A11); residues 50-66 of        SEQ ID NO:174 (CDR-H2 VH.2a 1A11); residues 99-107 of SEQ ID        NO:174 (CDR-H3 VH.2a 1A11); residues 24-34 of SEQ ID NO:175        (CDR-L1 VL.1 1A11); residues 50-56 of SEQ ID NO:175 (CDR-L2 VL.1        1A11); residues 89-97 of SEQ ID NO:175 (CDR-L3 VL.1 1A11);        residues 24-34 of SEQ ID NO:176 (CDR-L1 VL.1a 1A11);    -   residues 50-56 of SEQ ID NO:176 (CDR-L2 VL.1a 1A11); residues        89-97 of SEQ ID NO:176 (CDR-L3 VL.1a 1A11); residues 24-34 of        SEQ ID NO:177 (CDR-L1 VL.1b 1A11); residues 50-56 of SEQ ID        NO:177 (CDR-L2 VL.1b 1A11); residues 89-97 of SEQ ID NO:177        (CDR-L3 VL.1b 1A11); residues 24-34 of SEQ ID NO:178 (CDR-L1        VL.2a 1A11); residues 50-56 of SEQ ID NO:178 (CDR-L2 VL.2a        1A11); residues 89-97 of SEQ ID NO:178 (CDR-L3 VL.2a 1A11);        residues 31-35 of SEQ ID NO:179 (CDR-H1 VH.1 38H12); residues        50-66 of SEQ ID NO:179 (CDR-H2 VH.1 38H12); residues 99-107 of        SEQ ID NO:179 (CDR-H3 VH.1 38H12); residues 31-35 of SEQ ID        NO:180 (CDR-H1 VH.1A 38H12);    -   residues 50-66 of SEQ ID NO:180 (CDR-H2 VH.1A 38H12); residues        99-107 of SEQ ID NO:180 (CDR-H3 VH.1A 38H12); residues 31-35 of        SEQ ID NO:181 (CDR-H1 VH.1b 38H12); residues 50-66 of SEQ ID        NO:181 (CDR-H2 VH.1b 38H12); residues 99-107 of SEQ ID NO:181        (CDR-H3 VH.1b 38H12); residues 31-35 of SEQ ID NO:182 (CDR-H1        VH.2a 38H12); residues 50-66 of SEQ ID NO:182 (CDR-H2 VH.2a        38H12); residues 99-107 of SEQ ID NO:182 (CDR-H3 VH.2a 38H12);        residues 24-34 of SEQ ID NO:183 (CDR-L1 VL.1 38H12);    -   residues 50-56 of SEQ ID NO:183 (CDR-L2 VL.1 38H12); residues        89-97 of SEQ ID NO:183 (CDR-L3 VL.1 38H12); residues 24-34 of        SEQ ID NO:184 (CDR-L1 VL.1a 38H12); residues 50-56 of SEQ ID        NO:184 (CDR-L2 VL.1a 38H12);    -   residues 89-97 of SEQ ID NO:184 (CDR-L3 VL.1a 38H12); residues        24-34 of SEQ ID NO:185 (CDR-L1 VL.1b 38H12); residues 50-56 of        SEQ ID NO:185 (CDR-L2 VL.1b 38H12); residues 89-97 of SEQ ID        NO:185 (CDR-L3 VL.1b 38H12); residues 24-34 of SEQ ID NO:186        (CDR-L1 VL.2a 38H12); residues 50-56 of SEQ ID NO:186 (CDR-L2        VL.2a 38H12); residues 89-97 of SEQ ID NO:186 (CDR-L3 VL.2a        38H12); residues 31-35 of SEQ ID NO:187 (CDR-H1 h1A11VH.1),        residues 50-66 of SEQ ID NO:187 (CDR-H2 h1A11VH.1); residues        99-107 of SEQ ID NO:187 (CDR-H3 h1A11VH.1); residues 31-35 of        SEQ ID NO:188 (CDR-H1 h1A11.A6), residues 50-66 of SEQ ID NO:188        (CDR-H2 h1A11.A6); residues 99-107 of SEQ ID NO:188 (CDR-H3        h1A11.A6); residues 31-35 of SEQ ID NO:189 (CDR-H1 h1A11.A8),        residues 50-66 of SEQ ID NO:189 (CDR-H2 h1A11.A8); residues        99-107 of SEQ ID NO:189 (CDR-H3 h1A11.A8); residues 31-35 of SEQ        ID NO:190 (CDR-H1 h1A11.C6), residues 50-66 of SEQ ID NO:190        (CDR-H2 h1A11.C6); residues 99-107 of SEQ ID NO:190 (CDR-H3        h1A11.C6); residues 31-35 of SEQ ID NO:191 (CDR-H1 h1A11.A11),        residues 50-66 of SEQ ID NO:191 (CDR-H2 h1A11.A11); residues        99-107 of SEQ ID NO:191 (CDR-H3 h1A11.A11); residues 31-35 of        SEQ ID NO:192 (CDR-H1 h1A11.B5), residues 50-66 of SEQ ID NO:192        (CDR-H2 h1A11.B5); residues 99-107 of SEQ ID NO:192 (CDR-H3        h1A11.B5); residues 31-35 of SEQ ID NO:193 (CDR-H1 h1A11.E12),        residues 50-66 of SEQ ID NO:193 (CDR-H2 h1A11.E12);    -   residues 99-107 of SEQ ID NO:193 (CDR-H3 h1A11.E12); residues        31-35 of SEQ ID NO:194 (CDR-H1 h1A11.G3), residues 50-66 of SEQ        ID NO:194 (CDR-H2 h1A11.G3); residues 99-107 of SEQ ID NO:194        (CDR-H3 h1A11.G3); residues 31-35 of SEQ ID NO:195 (CDR-H1        h1A11.F5), residues 50-66 of SEQ ID NO:195 (CDR-H2 h1A11.F5);        residues 99-107 of SEQ ID NO:195 (CDR-H3 h1A11.F5);    -   residues 31-35 of SEQ ID NO:196 (CDR-H1 h1A11.H2), residues        50-66 of SEQ ID NO:196 (CDR-H2 h1A11.H2); residues 99-107 of SEQ        ID NO:196 (CDR-H3 h1A11.H2); residues 24-34 of SEQ ID NO:197        (CDR-L1 h1A11VL.1), residues 50-56 of SEQ ID NO:197 (CDR-L2        h1A11VL.1); residues 89-97 of SEQ ID NO:197 (CDR-L3 h1A11VL.1);        residues 24-34 of SEQ ID NO:198 (CDR-L1 h1A11.A2), residues        50-56 of SEQ ID NO:198 (CDR-L2 h1A11.A2); residues 89-97 of SEQ        ID NO:198 (CDR-L3 h1A11.A2); residues 24-34 of SEQ ID NO:199        (CDR-L1 h1A11.A12), residues 50-56 of SEQ ID NO:199 (CDR-L2        h1A11.A12);    -   residues 89-97 of SEQ ID NO:199 (CDR-L3 h1A11.A12); residues        24-34 of SEQ ID NO:200 (CDR-L1 h1A11.A7), residues 50-56 of SEQ        ID NO:200 (CDR-L2 h1A11.A7); residues 89-97 of SEQ ID NO:200        (CDR-L3 h1A11.A7); residues 24-34 of SEQ ID NO:201 (CDR-L1        h1A11.B4), residues 50-56 of SEQ ID NO:201 (CDR-L2 h1A11.B4);        residues 89-97 of SEQ ID NO:201 (CDR-L3 h1A11.B4);    -   residues 24-34 of SEQ ID NO:202 (CDR-L1 h1A11.B5), residues        50-56 of SEQ ID NO:202 (CDR-L2 h1A11.B5); residues 89-97 of SEQ        ID NO:202 (CDR-L3 h1A11.B5); residues 24-34 of SEQ ID NO:203        (CDR-L1 h1A11.E12), residues 50-56 of SEQ ID NO:203 (CDR-L2        h1A11.E12); and residues 89-97 of SEQ ID NO:203 (CDR-L3        h1A11.E12).

In an embodiment, a DLL4 binding protein of the invention comprises atleast three CDRs described herein (above or below). In a non-limitingexample, a DLL4 binding protein of the invention comprises three CDRsdescribed herein, wherein the three CDRs are a CDR-H1, a CDR-H2, and aCDR-H3 as described herein. In another non-limiting example, a DLL4binding protein of the invention comprising three CDRs described herein,wherein the three CDRs are a CDR-L1, a CDR-L2, and a CDR-L3 as describedherein.

In an embodiment, a DLL4 binding protein of the invention comprises oneor more CDRs described herein (above or below), such as one, two, three,four, five, or six CDRs described herein. In a preferred embodiment, aDLL4 binding protein according to the invention comprises six CDRsdescribed herein, e.g., a CDR-H1, a CDR-H2, a CDR-H3, a CDR-L1, aCDR-L2, and a CDR-L3 as described herein.

In another embodiment, a DLL4 binding protein of the invention comprisesthree CDRs selected from a set of variable domain CDRs, wherein the setof variable domain CDRs is selected from the group of variable domainCDR sets consisting of:

VH 38H12 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:157

CDR-H2: residues 50-66 of SEQ ID NO:157

CDR-H3 residues 99-107 of SEQ ID NO:157

VL 38H12 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:158

CDR-L2: residues 50-56 of SEQ ID NO:158

CDR-L3: residues 89-97 of SEQ ID NO:158

VH 1A11 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:159

CDR-H2: residues 50-66 of SEQ ID NO:159

CDR-H3: residues 99-107 of SEQ ID NO:159

VL 1A11 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:160

CDR-L2: residues 50-56 of SEQ ID NO:160

CDR-L3: residues 89-97 of SEQ ID NO:160

VH 37D10 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:161

CDR-H2: residues 50-68 of SEQ ID NO:161

CDR-H3: residues 101-111 of SEQ ID NO:161

VL 37D10 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:162

CDR-L2: residues 50-56 of SEQ ID NO:162

CDR-L3: residues 89-97 of SEQ ID NO:162

VH 32C7 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:163

CDR-H2: residues 50-66 of SEQ ID NO:163

CDR-H3: residues 99-105 of SEQ ID NO:163

VL 32C7 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:164

CDR-L2: residues 50-56 of SEQ ID NO:164

CDR-L3: residues 89-98 of SEQ ID NO:164

VH 14G1 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:165

CDR-H2: residues 50-66 of SEQ ID NO:165

CDR-H3: residues 99-105 of SEQ ID NO:165

VL 14G1 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:166

CDR-L2: residues 50-56 of SEQ ID NO:166

CDR-L3: residues 89-97 of SEQ ID NO:166

VH 14A11 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:167

CDR-H2: residues 50-66 of SEQ ID NO:167

CDR-H3: residues 99-110 of SEQ ID NO:167

VL 14A11 CDR Set

CDR-L1: residues 23-37 of SEQ ID NO:168

CDR-L2: residues 53-59 of SEQ ID NO:168

CDR-L3: residues 92-100 of SEQ ID NO:168

VH 15D6 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:169

CDR-H2: residues 50-66 of SEQ ID NO:169

CDR-H3: residues 99-110 of SEQ ID NO:169

VL 15D6 CDR Set

CDR-L1: residues 23-37 of SEQ ID NO:170

CDR-L2: residues 53-59 of SEQ ID NO:170

CDR-L3: residues 92-100 of SEQ ID NO:170

VH VH.1 1A11 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:171

CDR-H2: residues 50-66 of SEQ ID NO:171

CDR-H3: residues 99-107 of SEQ ID NO:171

VH VH.1a 1A11 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:172

CDR-H2: residues 50-66 of SEQ ID NO:172

CDR-H3: residues 99-107 of SEQ ID NO:172

VH VH.1b 1A11 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:173

CDR-H2: residues 50-66 of SEQ ID NO:173

CDR-H3: residues 99-107 of SEQ ID NO:173

VH VH.2a 1A11 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:174

CDR-H2: residues 50-66 of SEQ ID NO:174

CDR-H3: residues 99-107 of SEQ ID NO:174

VL VL.1 1A11 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:175

CDR-L2: residues 50-56 of SEQ ID NO:175

CDR-L3: residues 89-97 of SEQ ID NO:175

VL VL.1a 1A11 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:176

CDR-L2: residues 50-56 of SEQ ID NO:176

CDR-L3: residues 89-97 of SEQ ID NO:176

VL VL.1b 1A11 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:177

CDR-L2: residues 50-56 of SEQ ID NO:177

CDR-L3: residues 89-97 of SEQ ID NO:177

VL VL.2a 1A11 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:178

CDR-L2: residues 50-56 of SEQ ID NO:178

CDR-L3: residues 89-97 of SEQ ID NO:178

VH VH.1 38H12 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:179

CDR-H2: residues 50-66 of SEQ ID NO:179

CDR-H3: residues 99-107 of SEQ ID NO:179

VH VH.1a 38H12 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:180

CDR-H2: residues 50-66 of SEQ ID NO:180

CDR-H3: residues 99-107 of SEQ ID NO:180

VH VH.1b 38H12 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:181

CDR-H2: residues 50-66 of SEQ ID NO:181

CDR-H3: residues 99-107 of SEQ ID NO:181

VH VH.2a 38H12 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:182

CDR-H2: residues 50-66 of SEQ ID NO:182

CDR-H3: residues 99-107 of SEQ ID NO:182

VL VL.1 38H12 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:183

CDR-L2: residues 50-56 of SEQ ID NO:183

CDR-L3: residues 89-97 of SEQ ID NO:183

VL VL.1a 38H12 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:184

CDR-L2: residues 50-56 of SEQ ID NO:184

CDR-L3: residues 89-97 of SEQ ID NO:184

VL VL.1b 38H12 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:185

CDR-L2: residues 50-56 of SEQ ID NO:185

CDR-L3: residues 89-97 of SEQ ID NO:185

VL VL.2a 38H12 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:186

CDR-L2: residues 50-56 of SEQ ID NO:186

CDR-L3: residues 89-97 of SEQ ID NO:186

VH hA11VH.1 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:187

CDR-H2: residues 50-66 of SEQ ID NO:187

CDR-H3: residues 99-107 of SEQ ID NO:187

VH hA11.A6 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:188

CDR-H2: residues 50-66 of SEQ ID NO:188

CDR-H3: residues 99-107 of SEQ ID NO:188

VH hA11.A8 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:189

CDR-H2: residues 50-66 of SEQ ID NO:189

CDR-H3: residues 99-107 of SEQ ID NO:189

VH hA11.C6 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:190

CDR-H2: residues 50-66 of SEQ ID NO:190

CDR-H3: residues 99-107 of SEQ ID NO:190

VH hA11.A11 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:191

CDR-H2: residues 50-66 of SEQ ID NO:191

CDR-H3: residues 99-107 of SEQ ID NO:191

VH hA11.B5 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:192

CDR-H2: residues 50-66 of SEQ ID NO:192

CDR-H3: residues 99-107 of SEQ ID NO:192

VH hA11.E12 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:193

CDR-H2: residues 50-66 of SEQ ID NO:193

CDR-H3: residues 99-107 of SEQ ID NO:193

VH hA11.G3 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:194

CDR-H2: residues 50-66 of SEQ ID NO:194

CDR-H3: residues 99-107 of SEQ ID NO:194

VH hA11.F5 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:195

CDR-H2: residues 50-66 of SEQ ID NO:195

CDR-H3: residues 99-107 of SEQ ID NO:195

VH hA11.H2 CDR Set

CDR-H1: residues 31-35 of SEQ ID NO:196

CDR-H2: residues 50-66 of SEQ ID NO:196

CDR-H3: residues 99-107 of SEQ ID NO:196

VL h1A11VL.1 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:197

CDR-L2: residues 50-56 of SEQ ID NO:197

CDR-L3: residues 89-97 of SEQ ID NO:197

VL h1A11.A2 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:198

CDR-L2: residues 50-56 of SEQ ID NO:198

CDR-L3: residues 89-97 of SEQ ID NO:198

VL h1A11.A12 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:199

CDR-L2: residues 50-56 of SEQ ID NO:199

CDR-L3: residues 89-97 of SEQ ID NO:199

VL h1A11.A7 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:200

CDR-L2: residues 50-56 of SEQ ID NO:200

CDR-L3: residues 89-97 of SEQ ID NO:200

VL h1A11.B4 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:201

CDR-L2: residues 50-56 of SEQ ID NO:201

CDR-L3: residues 89-97 of SEQ ID NO:201

VL h1A11.B5 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:202

CDR-L2: residues 50-56 of SEQ ID NO:202

CDR-L3: residues 89-97 of SEQ ID NO:202 and

VL h1A11.E12 CDR Set

CDR-L1: residues 24-34 of SEQ ID NO:203

CDR-L2: residues 50-56 of SEQ ID NO:203

CDR-L3: residues 89-97 of SEQ ID NO:203

In an embodiment, a DLL4 binding protein comprises CDRs from at leasttwo sets of variable domain CDRs in the group above.

In another embodiment, a DLL4 binding protein of the invention comprisesthree CDRs selected from any VH set of three CDRs in the group above andthree CDRs selected from any VL set of three CDRs in the group above.

In still another embodiment, a DLL4 binding protein of the inventioncomprises a VH set of three CDRs as described above and a VL set ofthree CDRs as described above from a pair of VH and VL sets of CDRsselected from the group consisting of:

VH 38H12 CDR Set and VL 38H12 CDR Set,

VH 1A11 CDR Set and VL 1A11 CDR Set,

VH 37D10 CDR Set and VL 37D10 CDR Set,

VH 32C7 CDR Set and VL 32C7 CDR Set,

VH 14G1 Set and VL 14G1 CDR Set,

VH 14A11 CDR Set and VL 14A11 CDR Set,

VH 15D6 CDR Set and VL 15D6 CDR Set,

VH VH.1 1A11 CDR Set and VL VL.1 1A11 CDR Set,

VH VH.1 1A11 CDR Set and VL VL.1a 1A11 CDR Set,

VH VH.1 1A11 CDR Set and VL VL.1b 1A11 CDR Set,

VH VH.1 1A11 CDR Set and VL VL.2a 1A11 CDR Set,

VH VH.1a 1A11 CDR Set and VL VL.1 1A11 CDR Set,

VH VH.1a 1A11 CDR Set and VL VL.1a 1A11 CDR Set,

VH VH.1a 1A11 CDR Set and VL VL.1b 1A11 CDR Set,

VH VH.1a 1A11 CDR Set and VL VL.2a 1A11 CDR Set,

VH VH.1b 1A11 CDR Set and VL VL.1 1A11 CDR Set,

VH VH.1b 1A11 CDR Set and VL VL.1a 1A11 CDR Set,

VH VH.1b 1A11 CDR Set and VL VL.1b 1A11 CDR Set,

VH VH.1b 1A11 CDR Set and VL VL.2a 1A11 CDR Set,

VH VH.2a 1A11 CDR Set and VL VL.1 1A11 CDR Set,

VH VH.2a 1A11 CDR Set and VL VL.1a 1A11 CDR Set,

VH VH.2a 1A11 CDR Set and VL VL.1b 1A11 CDR Set,

VH VH.2a 1A11 CDR Set and VL VL.2a 1A11 CDR Set,

VH VH.1 38H12 CDR Set and VL VL.1 38H12 CDR Set,

VH VH.1 38H12 CDR Set and VL VL.1a 38H12 CDR Set,

VH VH.1 38H12 CDR Set and VL VL.1b 38H12 CDR Set,

VH VH.1 38H12 CDR Set and VL VL.2a 38H12 CDR Set,

VH VH.1a 38H12 CDR Set and VL VL.1 38H12 CDR Set,

VH VH.1a 38H12 CDR Set and VL VL.1a 38H12 CDR Set,

VH VH.1a 38H12 CDR Set and VL VL.1b 38H12 CDR Set,

VH VH.1a 38H12 CDR Set and VL VL.2a 38H12 CDR Set,

VH VH.1b 38H12 CDR Set and VL VL.1 38H12 CDR Set,

VH VH.1b 38H12 CDR Set and VL VL.1a 38H12 CDR Set,

VH VH.1b 38H12 CDR Set and VL VL.1b 38H12 CDR Set,

VH VH.1b 38H12 CDR Set and VL VL.2a 38H12 CDR Set,

VH VH.2a 38H12 CDR Set and VL VL.1 38H12 CDR Set,

VH VH.2a 38H12 CDR Set and VL VL.1a 38H12 CDR Set,

VH VH.2a 38H12 CDR Set and VL VL.1b 38H12 CDR Set,

VH VH.2a 38H12 CDR Set and VL VL.2a 38H12 CDR Set,

VH h1A11.A6 CDR Set and VL h1A11VL.1 CDR Set,

VH h1A11.C6 CDR Set and VL h1A11VL.1 CDR Set,

VH h1A11.A11 CDR Set and VL h1A11VL.1 CDR Set,

VH h1A11.A8 CDR Set and VL h1A11VL.1 CDR Set,

VH h1A11VH.1 CDR Set and VL h1A11.B4 CDR Set,

VH h1A11VH.1 CDR Set and VL h1A11.A7 CDR Set,

VH h1A11VH.1 CDR Set and VL h1A11.A12 CDR Set,

VH h1A11VH.1 CDR Set and VL h1A11.A2 CDR Set,

VH h1A11.B5 CDR Set and VL h1A11.B5 CDR Set,

VH h1A11.E12 CDR Set and VL h1A11.E12 CDR Set,

VH h1A11.G3 CDR Set and VL h1A11.E12 CDR Set,

VH h1A11.F5 CDR Set and VL h1A11.E12 CDR Set, and

VH h1A11.H2 CDR Set and VL h1A11.E12 CDR Set.

In a preferred embodiment, a DLL4 binding protein possess a DLL4 antigenbinding domain (or binding site) comprising six CDRs, wherein a CDR-H1,CDR-H2, and CDR-H3 are located in a heavy chain variable region (VH) anda CDR-L1, CDR-L2, and CDR-L3 are located in a light chain variableregion (VL), and wherein association of the VH and VL regions form afunctional DLL4 antigen binding domain of the DLL4 binding protein. In afurther non-limiting example of this embodiment, a DLL4 binding proteinthat possesses two DLL4 antigen binding domains comprises two sets of VHand VL regions and therefore comprises twelve CDRs.

In another embodiment, a DLL4 binding protein possesses a DLL4 antigenbinding domain comprising six CDRs, wherein a CDR-H1, CDR-H2, and CDR-H3are located in a heavy chain variable region (VH) and a CDR-L1, CDR-L2,and CDR-L3 are located in a light chain variable region (VL) and whereinthe remaining sequences in each variable region constitute a framework(FR) region such that each CDR is positioned between two FR regionsequences for a total of four FR sequences, i.e., FR1, FR2, FR3, andFR4. In this embodiment, the arrangement of FR and CDR sequences in avariable region is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In this embodiment, abinding domain formed by association of a VH region and a VL regioncomprises eight FR sequences and six CDRs.

DLL4 binding proteins of the invention include CDR-grafted antibodies,wherein one or more CDRs of VH and/or VL regions of an antibody of onespecies (donor species) are grafted into and replace the correspondingCDRs of the VH and/or VL of an antibody of another (acceptor) speciesusing recombinant techniques available in the art. An example of a donorspecies is a rat anti-human DLL4 monoclonal antibody described hereinand an example of an acceptor species is a human immunoglobulin gamma(IgG) molecule, wherein the human FR sequences of the VH and VL regionsof the human IgG molecule are human acceptor framework sequences thatreceive the grafted in CDRs from the donor rat monoclonal antibody. Thehuman acceptor framework sequences of the resulting CDR-grafted antibodymay be further mutated to improve one or more properties of theCDR-grafted antibody. By way of non-limiting examples, one or moreresidues of one or more FR sequences of a CDR-grafted antibody may bemutated to improve DLL4 binding affinity or to lower immunogenicity ofthe CDR-grafted antibody in a human subject.

In an embodiment of the invention, a DLL4 binding protein comprising oneor more CDRs described above further comprises a human acceptorframework sequence. Preferably, a DLL4 binding protein comprises one ormore (e.g., one, two, three, four, five, six, seven, or eight) humanacceptor framework sequences.

A human acceptor framework sequence present in a DLL4 binding protein ofthe invention may comprise one or more amino acid residues that havebeen back mutated to one or more corresponding amino acid residuespresent in a rat monoclonal antibody that binds DLL4 and/or that havebeen mutated to one or more amino acid residues that reduce or eliminatea site(s) for an undesirable reaction, for example, to reduce oreliminate a site for undesired glycosylation and/or a site for undesiredN-terminal pyroglutamate formation and/or a site for potentially reducedimmunogenicity risk.

In an embodiment, a DLL4 binding protein comprising one or more CDRsdescribed above further comprises one or more (e.g., any one, two,three, four, five, six, seven, or eight) of the human acceptor frameworksequences selected from the group of human acceptor framework sequencesin Tables 3 and 4, below. One or more human acceptor framework sequencesfrom Tables 3 and 4 present in a DLL4 binding protein of the inventionmay further comprise one or more amino acid residues that have been backmutated to one or more corresponding amino acid residues present in arat monoclonal antibody that binds DLL4 and/or that have been mutated toone or more amino acids that reduce or eliminate a site(s) for anundesirable reaction, for example, to reduce or eliminate a site forundesired glycosylation and/or a site for undesired N-terminalpyroglutamate formation and/or a site for potentially reducedimmunogenicity risk.

In another embodiment, the invention provides a DLL4 binding proteincomprising one or more CDRs described above, wherein the binding proteinalso comprises one or more (e.g., any one, two, three, four, five, six,seven, or eight per binding domain) of the human acceptor frameworksequences selected from any framework sequence present in a variableregion sequence selected from the group consisting of:

SEQ ID NO: 171 SEQ ID NO: 180 SEQ ID NO: 188 SEQ ID NO: 196 VH.1 1A11VH.1a 38H12 VH h1A11.A6 VH h1A11.H2 SEQ ID NO: 172 SEQ ID NO: 181 SEQ IDNO: 189 SEQ ID NO: 197 VH.1a 1A11 VH.1b 38H12 VH h1A11.A8 VL h1A11VL.1SEQ ID NO: 173 SEQ ID NO: 182 SEQ ID NO: 190 SEQ ID NO: 198 VH.1b 1A11VH.2a 38H12 VH h1A11.C6 VL h1A11.A2 SEQ ID NO: 174 SEQ ID NO: 183 SEQ IDNO: 191 SEQ ID NO: 199 VH.2a 1A11 VL.1 38H12 VH h1A11.A11 VL h1A11.A12SEQ ID NO: 175 SEQ ID NO: 184 SEQ ID NO: 192 SEQ ID NO: 200 VL.1 1A11VL.1a VH h1A11.B5 VL h1A11.A7 SEQ ID NO: 176 SEQ ID NO: 185 SEQ ID NO:193 SEQ ID NO: 201 VL.1a 1A11 VL.1b VH h1A11.E12 VL h1A11.B4 SEQ ID NO:177 SEQ ID NO: 186 SEQ ID NO: 194 SEQ ID NO: 202 VL.1b 1A11 VL.2a VHh1A11.G3 VL h1A11.B5 SEQ ID NO: 178 SEQ ID NO: 187 SEQ ID NO: 195 SEQ IDNO: 203 VL.2a 1A11 VH h1A11VH.1 VH h1A11.F5 VL h1A11.E12 SEQ ID NO: 179VH.1 38H12

In yet another embodiment of the invention, a DLL4 binding proteinfurther comprises one or more (e.g., any one, two, three, four, five,six, seven, or eight) acceptor framework sequences selected from thegroup consisting of:

-   -   heavy chain framework-1 (H-FR1):    -   E-V-Q-L-V-E-S-G-G-G-L-V-Q-P-G-G-S-L-R-L-S-C-A-A-S-G-F-T-F-X₃₀        (SEQ ID NO:143), wherein X₃₀ is S, R, or G;    -   heavy chain framework-2 (H-FR2): W—V-R-Q-A-P-G-K-G-L-E-W-V-A        (SEQ ID NO:144);    -   heavy chain framework-3 (H-FR3):    -   R-F-T-I-S-R-D-N-A-K-X₁₁-S-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-X₃₁-R        (SEQ ID NO:145), wherein;    -   X₁₁ is N or S; and    -   X₃₁ is A or S;    -   heavy chain framework-4 (H-FR4): W-G-Q-G-T-L-V-T-V-S-S(SEQ ID        NO:146);    -   light chain framework-1 (L-FR1):    -   D-I-Q-M-T-Q-S—P-S-S-L-S-A-S-V-G-D-R-V-T-I-T-C (SEQ ID NO:147);    -   light chain framework-2 (L-FR2): (SEQ ID NO:148), wherein;    -   X₉ is A or S; and    -   X₁₅ is F or Y;    -   light chain framework-3 (L-FR3):    -   G-V-P-S-R-F-S-G-S-G-S-G-T-D-X₁₅-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C        (SEQ ID NO:149), wherein;    -   X₁₅ is F or S; and    -   light chain framework-4 (L-FR4): F-G-Q-G-T-K-L-E-I-K (SEQ ID        NO:150).

In another embodiment, a DLL4 binding protein comprising one or moreCDRs described above also comprises a human acceptor framework sequencedescribed above wherein the human acceptor framework sequence comprisesat least one framework region amino acid substitution at a key residue,wherein the key residue is selected from the group consisting of aresidue adjacent to a CDR, a glycosylation site residue, a rare residue,a residue capable of interacting with human DLL4, a residue capable ofinteracting with a CDR, a canonical residue, a contact residue betweenheavy chain variable region and light chain variable region, a residuewithin a Vernier zone, and a residue in a region that overlaps between aChothia-defined variable heavy chain CDR1 and a Kabat-defined firstheavy chain framework.

In another embodiment, a human acceptor framework sequence of a DLL4binding protein described herein comprises at least one framework regionamino acid substitution, wherein the amino acid sequence of theframework is at least 65% identical to a sequence of a human germlineacceptor framework and comprises at least 70 amino acid residuesidentical to the human germline acceptor framework. In anotherembodiment, a DLL4 binding protein of the invention comprises aconsensus human variable domain sequence.

In an embodiment, the invention provides a DLL4 binding proteincomprises a human acceptor framework sequence, wherein the bindingprotein comprises at least one variable domain having an amino acidsequence selected from the group consisting of:

SEQ ID NO: 171 SEQ ID NO: 188 VH VH.1 1A11 VH h1A11.A6 SEQ ID NO: 172SEQ ID NO: 189 VH VH.1a 1A11 VH h1A11.A8 SEQ ID NO: 173 SEQ ID NO: 190VH VH.1b 1A11 VH h1A11.C6 SEQ ID NO: 174 SEQ ID NO: 191 VH VH.2a 1A11 VHh1A11.A11 SEQ ID NO: 175 SEQ ID NO: 192 VL VL.1 1A11 VH h1A11.B5 SEQ IDNO: 176 SEQ ID NO: 193 VL VL.1a 1A11 VH h1A11.E12 SEQ ID NO: 177 SEQ IDNO: 194 VL VL.1b VH h1A11.G3 SEQ ID NO: 178 SEQ ID NO: 195 VL VL.2a 1A11VH h1A11.F5 SEQ ID NO: 179 SEQ ID NO: 196 VH VH.1 38H12 VH h1A11.H2 SEQID NO: 180 SEQ ID NO: 197 VH VH.1a 38H12 VL h1A11VL.1 SEQ ID NO: 181 SEQID NO: 198 VH VH.1b 38H12 VL h1A11.A2 SEQ ID NO: 182 SEQ ID NO: 199 VHVH.2a 38H12 VL h1A11.A12 SEQ ID NO: 183 SEQ ID NO: 200 VL VL.1 38H12 VLh1A11.A7 SEQ ID NO: 184 SEQ ID NO: 201 VL VL.1a 38H12 VL h1A11.B4 SEQ IDNO: 185 SEQ ID NO: 202 VL VL.1b 38H12 VL h1A11.B5 SEQ ID NO: 186 SEQ IDNO: 203 VL VL.2a 38H12 VL h1A11.E12 SEQ ID NO: 187 VH h1A11VH.1

In another embodiment, a DLL4 binding protein of the invention comprisestwo or more variable domains described above. In a preferred embodiment,a DLL4 binding protein of the invention comprises two variable domains,wherein the two variable domains have amino acid sequences selected fromthe group consisting of:

SEQ ID NO: 171 and SEQ ID SEQ ID NO: 180 and SEQ ID NO: 175 NO: 186 VH.1and VL.1 1A11 VH.1a and VL.2a (Table 12) (Table 16) SEQ ID NO: 171 andSEQ ID SEQ ID NO: 181 and SEQ ID NO: 176 NO: 183 VH.1 and VL.1a 1A11VH.1b and VL.1 (Table 12) (Table 16) SEQ ID NO: 171 and SEQ ID SEQ IDNO: 181 and SEQ ID NO: 177 NO: 184 VH.1 and VL.1b 1A11 VH.1b and VL.1a(Table 12) (Table 16) SEQ ID NO: 171 and SEQ ID SEQ ID NO: 181 and SEQID NO: 178 NO: 185 VH.1 and VL.2a 1A11 VH.1b and VL.1b (Table 12) (Table16) SEQ ID NO: 172 and SEQ ID SEQ ID NO: 181 and SEQ ID NO: 175 NO: 186VH.1a and VL.1 1A11 (Table 12) VH.1b and VL.2a (Table 16) SEQ ID NO: 172and SEQ ID SEQ ID NO: 182 and SEQ ID NO: 176 NO: 183 VH.1a and VL.1a1A11 (Table 12) VH.2a and VL.1 (Table 16) SEQ ID NO: 172 and SEQ ID SEQID NO: 182 and SEQ ID NO: 177 NO: 184 VH.1a and VL.1b 1A11 VH.2a andVL.1a (Table 12) (Table 16) SEQ ID NO: 172 and SEQ ID SEQ ID NO: 182 andSEQ ID NO: 178 NO: 185 VH.1a and VL.2a 1A11 VH.2a and VL.1b (Table 12)(Table 16) SEQ ID NO: 173 and SEQ ID SEQ ID NO: 182 and SEQ ID NO: 175NO: 186 VH.1b and VL.1 1A11 VH.2a and VL.2a (Table 12) (Table 16) SEQ IDNO: 173 and SEQ ID SEQ ID NO: 188 and SEQ ID NO: 176 NO: 197 VH.1b andVL.1a 1A11 h1A11.A6 VH and h1A11VL.1 (Table 12) Tables 20/21 SEQ ID NO:173 and SEQ ID SEQ ID NO: 190 and SEQ ID NO: 177 NO: 197 VH.1b and VL.1b1A11 h1A11.C6 VH and h1A11VL.1 (Table 12) Tables 20/21 SEQ ID NO: 173and SEQ ID SEQ ID NO: 191 and SEQ ID NO: 178 NO: 197 VH.1b and VL.2a1A11 h1A11.All VH and h1A11VL.1 (Table 12) Tables 20/21 SEQ ID NO: 174and SEQ ID SEQ ID NO: 189 and SEQ ID NO: 175 NO: 197 VH.2a and VL.1 1A11h1A11.A8 VH and h1A11 VL.1 (Table 12) Tables 20/21 SEQ ID NO: 174 andSEQ ID SEQ ID NO: 1878 and SEQ ID NO: 176 NO: 201 VH.2a and VL.1a 1A11h1A11VH.1 and h1A11.B4 VL (Table 12) Tables 20/21 SEQ ID NO: 174 and SEQID SEQ ID NO: 187 and SEQ ID NO: 177 NO: 200 VH.2a and VL.1b 1A11h1A11VH.1 and h1All.A7 VL (Table 12) Tables 20/21 SEQ ID NO: 174 and SEQID SEQ ID NO: 187 and SEQ ID NO: 178 NO: 199 VH.2a and VL.2a 1A11h1A11VH.1 and h1All.A12 VL (Table 12) Tables 20/21 SEQ ID NO: 179 andSEQ ID SEQ ID NO: 187 and SEQ ID NO: 183 NO: 198 VH.1 and VL.1 38H12h1A11VH.1 VH and h1All.A2 VL (Table 16) Tables 20/21 SEQ ID NO: 179 andSEQ ID SEQ ID NO: 192 and SEQ ID NO: 184 NO: 202 VH.1 and VL.1a 38H12h1A11.B5 VH and h1All.B5 VL (Table 16) Tables 20/21 SEQ ID NO: 179 andSEQ ID SEQ ID NO: 193 and SEQ ID NO: 185 NO: 203 VH.1 and VL.1b 38H12h1A11.E12 VH and h1All.E12 VL (Table 16) Tables 20/21 SEQ ID NO: 179 andSEQ ID SEQ ID NO: 194 and SEQ ID NO: 186 NO: 203 VH.1 and VL.2a 38H12h1A11.G3 VH and h1All.E12 VL (Table 16) Tables 20/21 SEQ ID NO: 180 andSEQ ID SEQ ID NO: 195 and SEQ ID NO: 183 NO: 203 VH.1a and VL.1 38H12h1A11.F5 VH and h1All.E12 VL (Table 16) Tables 20/21 SEQ ID NO: 180 andSEQ ID SEQ ID NO: 196 and SEQ ID NO: 184 NO: 203 VH.1a and VL.1a 38H12h1A11.H2 VH and h1All.E12 VL (Table 16) Tables 20/21 SEQ ID NO: 180 andSEQ ID NO: 185 VH.1a and VL.1b 38H12 (Table 16)

In an embodiment, a DLL4 binding protein of the invention comprises atleast one variable domain having an amino acid sequence selected fromthe group consisting of:

SEQ ID NO: 157 SEQ ID NO: 181 VH 38H12 VH VH.1b 38H12 SEQ ID NO: 158 SEQID NO: 182 VL 38H12 VH VH.2a 38H12 SEQ ID NO: 159 SEQ ID NO: 182 VH 1A11VL VL.1 38H12 SEQ ID NO: 160 SEQ ID NO: 184 VL 1A11 VL VL.1a 38H12 SEQID NO: 161 SEQ ID NO: 185 VH 37D10 VL VL.1b 38H12 SEQ ID NO: 162 SEQ IDNO: 186 VL 37D10 VL VL.2a 38H12 SEQ ID NO: 163 SEQ ID NO: 187 VH 32C7 VHh1A11VH.1 SEQ ID NO: 164 SEQ ID NO: 188 VL 32C7 VH h1A11.A6 SEQ ID NO:165 SEQ ID NO: 189 VH 14G1 VH h1A11.A8 SEQ ID NO: 166 SEQ ID NO: 190 VL14G1 VH h1A11.C6 SEQ ID NO: 167 SEQ ID NO: 191 VH 14A11 VH h1A11.A11 SEQID NO: 168 SEQ ID NO: 192 VL 14A11 VH h1A11.B5 SEQ ID NO: 169 SEQ ID NO:193 VH 15D6 VH h1A11.E12 SEQ ID NO: 170 SEQ ID NO: 194 VL 15D6 VHh1A11.G3 SEQ ID NO: 171 SEQ ID NO: 195 VH VH.1 1A11 VH h1A11.F5 SEQ IDNO: 172 SEQ ID NO: 196 VH VH.1a 1A11 VH h1A11.H2 SEQ ID NO: 173 SEQ IDNO: 197 VH VH.1b 1A11 VL h1A11VL.1 SEQ ID NO: 174 SEQ ID NO: 198 VHVH.2a 1A11 VL h1A11.A2 SEQ ID NO: 175 SEQ ID NO: 199 VL VL.1 1A11 VLh1A11.A12 SEQ ID NO: 176 SEQ ID NO: 200 VL VL.1a 1A11 VL h1A11.A7 SEQ IDNO: 177 SEQ ID NO: 201 VL VL.1b VL h1A11.B4 SEQ ID NO: 178 SEQ ID NO:202 VL VL.2a 1A11 VL h1A11.B5 SEQ ID NO: 179 SEQ ID NO: 203 VH VH.138H12 VL h1A11.E12 SEQ ID NO: 180 VH VH.1a 38H12

In another embodiment, a DLL4 binding protein of the invention comprisestwo variable domains, wherein the two variable domains have the aminoacid sequences selected from the group consisting of:

SEQ ID NO: 157 and SEQ ID SEQ ID NO: 179 and SEQ ID NO: 158 NO: 18638H12 VH.1 and VL.2a 38H12 (Table 16) SEQ ID NO: 159 and SEQ ID SEQ IDNO: 180 and SEQ ID NO: 160 NO: 183 1A11 VH.1a and VL.1 38H12 (Table 16)SEQ ID NO: 161 and SEQ ID SEQ ID NO: 180 and SEQ ID NO: 162 NO: 18437D10 VH.1a and VL.1a 38H12 (Table 16) SEQ ID NO: 163 and SEQ ID SEQ IDNO: 180 and SEQ ID NO: 164 NO: 185 32C7 VH.1a and VL.1b 38H12 (Table 16)SEQ ID NO: 165 and SEQ ID SEQ ID NO: 180 and SEQ ID NO: 166 NO: 186 14G1VH.1a and VL.2a (Table 16) SEQ ID NO: 167 and SEQ ID SEQ ID NO: 181 andSEQ ID NO: 168 NO: 183 14A11 VH.1b and VL.1 (Table 16) SEQ ID NO: 169and SEQ ID SEQ ID NO: 181 and SEQ ID NO: 170 NO: 184 15D6 VH.1b andVL.1a (Table 16) SEQ ID NO: 171 and SEQ ID SEQ ID NO: 181 and SEQ ID NO:175 NO: 185 VH.1 and VL.1 1A11 VH.1b and VL.1b (Table 11) (Table 16) SEQID NO: 171 and SEQ ID SEQ ID NO: 181 and SEQ ID NO: 176 NO: 186 VH.1 andVL.1a 1A11 VH.1b and VL.2a (Table 11) (Table 16) SEQ ID NO: 171 and SEQID SEQ ID NO: 182 and SEQ ID NO: 177 NO: 183 VH.1 and VL.1b 1A11 VH.2aand VL.1 (Table 11) (Table 16) SEQ ID NO: 171 and SEQ ID SEQ ID NO: 182and SEQ ID NO: 178 NO: 184 VH.1 and VL.2a VH.2a and VL.1a (Table 11)(Table 16) SEQ ID NO: 172 and SEQ ID SEQ ID NO: 182 and SEQ ID NO: 175NO: 185 VH.1a and VL.1 VH.2a and VL.1b (Table 11) (Table 16) SEQ ID NO:172 and SEQ ID SEQ ID NO: 182 and SEQ ID NO: 176 NO: 186 VH.1a and VL.1aVH.2a and VL.2a (Table 11) (Table 16) SEQ ID NO: 172 and SEQ ID SEQ IDNO: 188 and SEQ ID NO: 177 NO: 197 VH.1a and VL.1b h1A11.A6 VH andh1A11VL.1 (Table 11) Tables 20/21 SEQ ID NO: 172 and SEQ ID SEQ ID NO:190 and SEQ ID NO: 178 NO: 197 VH.1a and VL.2a h1A11.C6 VH and h1A11VL.1(Table 11) Tables 20/21 SEQ ID NO: 173 and SEQ ID SEQ ID NO: 191 and SEQID NO: 175 NO: 197 VH.1b and VL.1 h1A11.All VH and h1A11VL.1 (Table 11)Tables 20/21 SEQ ID NO: 173 and SEQ ID SEQ ID NO: 189 and SEQ ID NO: 176NO: 197 VH.1b and VL.1a h1A11.A8 VH and h1A11 VL.1 (Table 11) Tables20/21 SEQ ID NO: 173 and SEQ ID SEQ ID NO: 187 and SEQ ID NO: 177 NO:201 VH.1b and VL.1b h1A11VH.1 and h1A11.B4 VL (Table 11) Tables 20/21SEQ ID NO: 173 and SEQ ID SEQ ID NO: 187 and SEQ ID NO: 178 NO: 200VH.1b and VL.2a h1A11VH.1 and h1All.A7 VL (Table 11) Tables 20/21 SEQ IDNO: 174 and SEQ ID SEQ ID NO: 187 and SEQ ID NO: 175 NO: 199 VH.2a andVL.1 h1A11VH.1 and h1All.A12 VL (Table 11) Tables 20/21 SEQ ID NO: 174and SEQ ID SEQ ID NO: 187 and SEQ ID NO: 176 NO: 198 VH.2a and VL.1ah1A11VH.1 VH and h1All.A2 VL (Table 11) Tables 20/21 SEQ ID NO: 174 andSEQ ID SEQ ID NO: 192 and SEQ ID NO: 177 NO: 202 VH.2a and VL.1bh1A11.B5 VH and h1All.B5 VL (Table 11) Tables 20/21 SEQ ID NO: 174 andSEQ ID SEQ ID NO: 193 and SEQ ID NO: 178 NO: 203 VH.2a and VL.2ah1A11.E12 VH and h1All.E12 VL (Table 11) Tables 20/21 SEQ ID NO: 179 andSEQ ID SEQ ID NO: 194 and SEQ ID NO: 183 NO: 203 VH.1 and VL.1 38H12h1A11.G3 VH and h1All.E12 VL (Table 16) Tables 20/21 SEQ ID NO: 179 andSEQ ID SEQ ID NO: 195 and SEQ ID NO: 184 NO: 203 VH.1 and VL.1a 38H12h1A11.F5 VH and h1All.E12 VL (Table 16) Tables 20/21 SEQ ID NO: 179 andSEQ ID SEQ ID NO: 196 and SEQ ID NO: 185 NO: 203 VH.1 and VL.1b 38H12h1A11.H2 VH and h1All.E12 VL (Table 16) Tables 20/21

In an embodiment, a DLL4 binding protein of the invention comprises twovariable domains having the amino acid sequences selected from the groupconsisting of:

-   -   SEQ ID NO:188 (h1A11.A6 VH) and SEQ ID NO:197 (h1A11VL.1),    -   SEQ ID NO:190 (h1A11.C6 VH) and SEQ ID NO:197 (h1A11VL.1), and    -   SEQ ID NO:191 (h1A11.A11 VH) and SEQ ID NO:197 (h1A11.VL.1).

In an embodiment, a DLL4 binding protein of the invention comprises twovariable domains having the amino acid sequences SEQ ID NO:181 (VH.1b38H12) and SEQ ID NO:185 (VL.1b 38H12).

According to the invention, variable heavy (VH) domains and variablelight (VL) domains of any of the DLL4 binding proteins described hereinmay also be shuffled using recombinant techniques available in the artto generate and select for additional DLL4 binding proteins thatcomprise various combinations of VH and VL domains described herein.

In an embodiment, a DLL4 binding protein according to the inventionbinds human DLL4 (hu DLL4) and at least one other species of DLL4. Morepreferably, a DLL4 binding protein described herein binds human DLL4 anda DLL4 selected from the group consisting of a cynomolgus monkey DLL4(cynomolgus DLL4, cyno DLL4), a mouse DLL4 (mu DLL4), a rat DLL4, andcombinations thereof.

In another embodiment, a DLL4 binding protein described herein iscapable of blocking DLL4 interaction with a Notch protein. Preferably,the Notch protein is selected from the group consisting of Notch-1,Notch-2, Notch-3, Notch-4, and combinations thereof.

In an embodiment, a DLL4 binding protein described herein is capable ofmodulating, inhibiting, or neutralizing one or more biological functionsof human DLL4. More preferably, a DLL4 binding protein of the inventionis capable of modulating, inhibiting, or neutralizing an activity of aDLL4 selected from the group consisting of a human DLL4, a cynomolgusDLL4, a monkey DLL4, a rat DLL4, and combinations thereof.

In a further embodiment, a DLL4 binding protein described herein iscapable of inhibiting VEGFR2 activity, VEGFR1 activity, or both VEGFR2and VEGFR1 activities.

In an embodiment, a DLL4 binding protein described herein is capable ofinhibiting normal angiogenesis.

In an embodiment, a DLL4 binding protein of the invention has an on rateconstant (K_(on)) to DLL4 of at least about 10²M⁻¹s⁻¹; at least about10³M⁻¹s⁻¹; at least about 10⁴M⁻¹s⁻¹; at least about 10⁵M⁻¹s⁻¹; or atleast about 10⁶M⁻¹s⁻¹, as measured by surface plasmon resonance.Preferably, the binding protein of the invention has an on rate constant(K_(on)) to DLL4 between 10²M⁻¹s⁻¹ to 10³M⁻¹s⁻¹; between 10³M⁻¹s⁻¹ to10⁴M⁻¹s⁻¹; between 10⁴M⁻¹s⁻¹ to 10⁵M⁻¹s⁻¹; or between 10⁵M⁻¹s⁻¹ to10⁶M⁻¹s⁻¹, as measured by surface plasmon resonance.

In another embodiment, a DLL4 binding protein of the invention has anoff rate constant (K_(off)) for DLL4 of at most about 10⁻³s⁻¹; at mostabout 10⁻⁴s⁻¹; at most about 10⁻⁵s⁻¹; or at most about 10⁻⁶s⁻¹, asmeasured by surface plasmon resonance. Preferably, the binding proteinof the invention has an off rate constant (K_(off)) to DLL4 of 10's⁻¹ to10⁻⁴s⁻¹; of 10's⁻¹ to 10's⁻¹; or of 10⁻⁵s⁻¹ to 10's⁻¹, as measured bysurface plasmon resonance.

In another embodiment, a DLL4 binding protein of the invention has adissociation constant (K_(D)) to DLL4 of at most about 10⁻⁷ M; at mostabout 10⁻⁸ M; at most about 10⁻⁹ M; at most about 10⁻¹⁰ M; at most about10⁻¹¹M; at most about 10⁻¹² M; or at most 10⁻¹³M. Preferably, thebinding protein of the invention has a dissociation constant (K_(D)) toDLL4 of 10⁻⁷ M to 10⁻⁸ M; of 10⁻⁸ M to 10⁻⁹M; of 10⁻⁹ M to 10⁻¹⁰ M; of10⁻¹⁰ to 10⁻¹¹M; of 10⁻¹¹ M to 10⁻¹² M; or of 10⁻¹² to M to 10⁻¹³M.

In an embodiment, the invention provides an antibody constructcomprising a DLL4 binding protein described above and a linkerpolypeptide or an immunoglobulin constant domain. In a preferredembodiment, an antibody construct according to the invention is selectedfrom the group consisting of: an immunoglobulin molecule, a monoclonalantibody, a chimeric antibody, a CDR-grafted antibody, a humanizedantibody, a Fab, a Fab′, a F(ab′)₂, an Fv, a disulfide linked Fv, ascFv, a single domain antibody, a diabody, a multispecific antibody, adual specific antibody, and a bispecific antibody.

In a preferred embodiment, an antibody construct of the inventioncomprises a heavy chain immunoglobulin constant domain selected from thegroup consisting of a human IgM constant domain, a human IgG1 constantdomain, a human IgG2 constant domain, a human IgG3 constant domain, ahuman IgG4 constant domain, a human IgE constant domain, and a human IgAconstant domain.

In another embodiment, an antibody construct of the invention comprisesan immunoglobulin constant region selected from the group consisting ofan immunoglobulin gamma-1 (IgG-1) heavy chain constant region (such asSEQ ID NO:3), a mutant IgG-1 heavy chain constant region (such as SEQ IDNO:4), an immunoglobulin kappa light chain constant region (such as SEQID NO:5), an immunoglobulin lambda light chain constant region (such asSEQ ID NO:6), and combinations thereof.

In another embodiment, an antibody construct is glycosylated.Preferably, the glycosylation is a human glycosylation pattern.

In an embodiment, the invention provides an antibody conjugatecomprising an antibody construct described herein conjugated to anagent. Preferably, the agent is selected from the group consisting of:an imaging agent, a therapeutic agent, a cytotoxic agent, and animmunoadhesion molecule. In a preferred embodiment, an imaging agents isselected from the group consisting of: a radiolabel, an enzyme, afluorescent label, a luminescent label, a bioluminescent label, amagnetic label, and biotin. More preferably the imaging agent is aradiolabel selected from the group consisting of: ³H, ¹⁴C, ³⁵S, ⁹⁰Y,⁹⁹Tc, ¹¹¹In, ¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, and ¹⁵³Sm. In a preferredembodiment, the therapeutic or cytotoxic agent is selected from thegroup consisting of: an anti-metabolite, an alkylating agent, anantibiotic, a growth factor, a cytokine, an anti-angiogenic agent, ananti-mitotic agent, an anthracycline, toxin, and an apoptotic agent.

In another embodiment, a DLL4 binding protein, an antibody construct, orantibody conjugate described above exists as a crystal. Preferably, thecrystal is a carrier-free pharmaceutical controlled release crystal. Inanother embodiment, such a crystallized binding protein, crystallizedantibody construct, or crystallized antibody conjugate has a greaterhalf life in vivo than its soluble counterpart. In a preferredembodiment, a crystallized binding protein, crystallized antibodyconstruct, or crystallized antibody conjugate retains the biologicalactivity of the soluble or non-crystal form of the binding protein,antibody construct, or antibody conjugate after crystallization.

In an embodiment, the invention provides an isolated nucleic acidencoding one or more amino acid sequences of a DLL4 binding protein(including any antibody construct or antibody conjugate) describedherein

In a preferred embodiment, the invention provides an isolated nucleicacid encoding a polypeptide selected from the group consisting of: apolypeptide comprising a heavy chain variable domain, wherein the heavychain variable domain comprises one or more of a CDR-H1, a CDR-H2, and aCDR-H3 as described above; a polypeptide comprising a light chainvariable domain, wherein the light chain variable domain comprises oneor more of a CDR-L1, a CDR-L2, and a CDR-L3 as described above; and acombination of both polypeptides.

One aspect of the invention pertains to an isolated nucleic acidencoding a DLL4 binding protein, an antibody construct, a DLL4 bindingantibody conjugate, or DLL4 binding portion thereof. Particularlypreferred is an isolated nucleic acid that encodes a polypeptideselected from the group consisting of: a polypeptide comprising a heavychain variable domain, wherein the heavy chain variable domain comprisesa CDR-H1, a CDR-H2, or a CDR-H3 described above; a polypeptidecomprising a light chain variable domain, wherein the light chainvariable domain comprises a CDR-L1, a CDR-L2, or a CDR-L3 as describedabove; or a combination of both polypeptides.

A further embodiment provides a vector comprising an isolated nucleicacid described herein. In a preferred embodiment, the vector is selectedfrom the group consisting of: pcDNA, pTT (Durocher et al., Nucl. AcidsRes., 30(2e9): 1-9 (2002)), pTT3 (pTT with additional multiple cloningsites), pEFBOS (Mizushima et al., Nucl. Acids. Res., 18 (17): 5322(1990)), pBV, pJV, and pBJ.

In another aspect of the invention there is provided a host celltransformed with the vector described above. The host cell can be aprokaryotic or eukaryotic cell. A preferred prokaryotic host cell isEscherichia coli. Preferably, the eukaryotic cell is selected from thegroup consisting of: a protist cell, an animal cell, a plant cell, and afungal cell. More preferably, the host cell is a mammalian cellincluding, but not limited to, CHO and COS cells. A preferred fungalcell is Saccharomyces cerevisiae. A preferred insect cell is an Sf9cell.

In another aspect of the invention there is provided a method ofproducing a binding protein that binds human DLL4 comprising the step ofculturing any one of the host cells described above in a culture mediumunder conditions sufficient to produce a binding protein that bindshuman DLL4.

One embodiment provides a composition for the release of a DLL4 bindingprotein according to the invention wherein the composition comprises aformulation that comprises a crystallized DLL4 binding protein, acrystallized antibody construct, or a crystallized antibody conjugate asdescribed above and an ingredient, and further at least one polymericcarrier. Preferably, the polymeric carrier is a polymer selected fromone or more of the group consisting of: poly (acrylic acid), poly(cyanoacrylates), poly (amino acids), poly (anhydrides), poly(depsipeptide), poly (esters), poly (lactic acid), poly(lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly(caprolactone), poly (dioxanone); poly (ethylene glycol), poly((hydroxypropyl) methacrylamide, poly [(organo)phosphazene], poly (orthoesters), poly (vinyl alcohol), poly (vinylpyrrolidone), maleicanhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,alginate, cellulose and cellulose derivatives, collagen, fibrin,gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans, sulfatedpolysaccharides, blends and copolymers thereof. Preferably, theingredient is selected from the group consisting of albumin, sucrose,trehalose, lactitol, gelatin, hydroxypropyl-β-cyclodextrin,methoxypolyethylene glycol and polyethylene glycol.

Another embodiment provides a method for treating a mammal comprisingthe step of administering to the mammal an effective amount of acomposition comprising a crystallized DLL4 binding protein, acrystallized antibody construct, or a crystallized antibody conjugatesdescribed above.

The invention also provides a pharmaceutical composition comprising aDLL4 binding protein as described above (including an antibody constructor an antibody conjugate as described above) and a pharmaceuticallyacceptable carrier. In a further embodiment, the pharmaceuticalcomposition comprises at least one additional agent. The additionalagent may be a therapeutic agent for treating a disorder in which DLL4is detrimental. Preferably, a pharmaceutical composition of theinvention comprises an additional agent selected from the groupconsisting of: a therapeutic agent; an imaging agent; an antineoplasticagent; a chemotherapeutic agent; an angiogenesis inhibitor; an anti-VEGFantibody; an anti-EGFR antibody; an anti-cMet antibody; an anti-ErbB3antibody; an anti-HER2 antibody; an anti-CD20 antibody; a VEGF-trapmolecule; a kinase inhibitor; a co-stimulation molecule blocker; ananti-B7.2 antibody; a CTLA4-Ig; an adhesion molecule blocker; an anti-Eselectin antibody; an anti-L selectin antibody; an anti-cytokineantibody or functional fragment thereof; an anti-IL-18 antibody; ananti-TNF antibody; anti-IL-6 antibody; methotrexate; a corticosteroid; acyclosporin; a rapamycin; FK506; a DNA alkylating agent; cisplatin;carboplatin; an anti-tubulin agent; paclitaxel; docetaxel; doxorubicin;gemcitabine; gemzar; an anthracycline; adriamycin; a topoisiomersase Iinhibitor; a topoisomerase II inhibitor; 5-fluorouracil (5-FU);leucovorin; irinotecan; a receptor tyrosine kinase inhibitor; anapoptosis inhibitor; a Bcl2/Bclx inhibitor; erlotinib; gefitinib; aCOX-2 inhibitor; celecoxib; cyclosporin; rapamycin; a detectable labelor reporter molecule; a TNF antagonist; an antirheumatic; a musclerelaxant; a narcotic; an analgesic; an anesthetic; a sedative; a localanesthetic; a neuromuscular blocker; an antimicrobial agent; anantipsoriatic agent; a corticosteroid; an anabolic steroid; anerythropoietin; an immunization; an immunoglobulin; an immunosuppressiveagent; a growth hormone; a hormone replacement drug; aradiopharmaceutical drug; an antidepressant; an antipsychotic drug; astimulant; an asthma medication; a beta agonist; an inhaled steroid; anepinephrine; an epinephrine analog thereof; a cytokine; and a cytokineantagonist.

In another aspect, the invention provides a method for inhibiting humanDLL4 activity comprising contacting human DLL4 with a binding proteindisclosed above such that human DLL4 is inhibited or neutralized. In arelated aspect, the invention provides a method for inhibiting DLL4activity in a human subject suffering from a disorder in which DLL4 isdetrimental, comprising administering to the human subject a bindingprotein disclosed above such that human DLL4 in the human subject isinhibited and treatment is achieved. Preferably, the disorder isselected from the group comprising primary and metastatic cancers,including carcinomas of breast, colon, rectum, lung, oropharynx,hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bileducts, small intestine, urinary tract (including kidney, bladder, andurothelium), female genital tract (including cervix, uterus, and ovariesas well as choriocarcinoma and gestational trophoblastic disease), malegenital tract (including prostate, seminal vesicles, testes, and germcell tumors), endocrine glands (including the thyroid, adrenal, andpituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas(including those arising from bone and soft tissues as well as Kaposi'ssarcoma), tumors of the brain, nerves, eyes, and meninges (includingastrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas,neuroblastomas, Schwannomas, and meningiomas), solid tumors arising fromhematopoietic malignancies such as leukemias and lymphomas (bothHodgkin's and non-Hodgkin's lymphomas), tumor metastases, ocularneovascularization (including diabetic blindness, retinopathies,age-induced macular degeneration and rubeosis), edema, rheumatoidarthritis, atherosclerotic plaques, refractory ascites, psoriasis,pancreatitis, polycystic ovarian disease (POD), endometriosis, uterinefibroids, benign prostate hypertrophy, T-cell acute lymphoblasticleukemia (T-ALL), cerebral autosomal dominant arteriopathy withsubcortical infarcts and leukoencephalopathy (CADASIL), multiplesclerosis (MS), tetralogy of Fallot (TOF), Alagille syndrome (AS),macular degeneration and age-related macular degeneration diseases, andother angiogenesis independent and dependent diseases characterized byaberrant DLL4 expression or activity.

In another aspect, the invention provides a method of treating a patientsuffering from a disorder in which human DLL4 is detrimental comprisingthe step of administering any one of the binding proteins disclosedabove before, concurrent, or after the administration of atherapeutically effective amount of a second agent. In a preferredembodiment, the second agent is selected from the group consisting of: aradiotherapeutic agent; an antineoplastic agent; a chemotherapeuticagent; a DNA alkylating agent; cisplatin; carboplatin; an anti-tubulinagent; paclitaxel; docetaxel; taxol; doxorubicin; gemcitabine; gemzar;an anthracycline; adriamycin; a topoisomerase I inhibitor; atopoisomerase II inhibitor; 5-fluorouracil (5-FU); leucovorin;irinotecan; a receptor tyrosine kinase inhibitor; an apoptosisinhibitor; a Bcl2/Bclx inhibitor; erlotinib; gefitinib; a COX-2inhibitor; celecoxib; a kinase inhibitor; an angiogenesis inhibitor; ananti-VEGF antibody; anti-EGFR antibody; an anti-cMet antibody; ananti-ErbB3 antibody; an anti-HER2 antibody; an anti-CD20 antibody;VEGF-Trap (aflibercept); a co-stimulation molecule blocker; an anti-B7.1antibody; an anti-B7.2 antibody; CTLA4-Ig; an adhesion molecule blocker;an anti-LFA-1 antibody; an anti-E selectin antibody; an anti-L selectinantibody; a small molecule inhibitor; an anti-cytokine antibody orfunctional fragment thereof; an anti-IL-18 antibody; an anti-TNFantibody; an anti-IL-6 antibody; an anti-cytokine receptor antibody;methotrexate; cyclosporin; rapamycin; FK506; a detectable label orreporter; a TNF antagonist; an antirheumatic; a muscle relaxant; anarcotic; a non-steroid anti-inflammatory drug (NSAID); an analgesic; ananesthetic; a sedative; a local anesthetic; a neuromuscular blocker; anantimicrobial agent; an antipsoriatic drug; a corticosteroid; ananabolic steroid; an erythropoietin; an immunization; an immunoglobulin;an immunosuppressive agent; a growth hormone; a hormone replacementdrug; a radiopharmaceutical drug; an antidepressant; an antipsychoticdrug; a stimulant; an asthma medication; a beta agonist; an inhaledsteroid; an epinephrine; an epinephrine analog; a cytokine; and acytokine antagonist.

In a preferred embodiment, the pharmaceutical compositions disclosedabove are administered to the subject by at least one mode selected fromthe group consisting of: parenteral, subcutaneous, intramuscular,intravenous, intraarterial, intraarticular, intrabronchial,intraabdominal, intracapsular, intracartilaginous, intracavitary,intracelial, intracerebellar, intracerebroventricular, intracolic,intracervical, intragastric, intrahepatic, intramyocardial, intraosteal,intrapelvic, intrapericardiac, intraperitoneal, intrapleural,intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal,intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical,bolus, vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

Another aspect of the invention provides at least one DLL4 anti-idiotypeantibody to at least one DLL4 binding protein of the present invention.The anti-idiotype antibody includes any protein or peptide containingmolecule that comprises at least a portion of an immunoglobulin moleculesuch as, but not limited to, at least one complementarily determiningregion (CDR) of a heavy or light chain or a ligand binding portionthereof, a heavy chain or light chain variable region, a heavy chain orlight chain constant region, a framework region, and any portionthereof, that can be incorporated into a binding protein of the presentinvention.

Any of a variety of immunodetection assay formats may be adapted toemploy a DLL4 binding protein of the invention to detect or measure DLL4in a mixture, solution, or biological sample. Such immunodetection assayformats include but are not limited to radioimmunoassay (RIA),immunoprecipitation, enzyme-linked immunosorbent assay (ELISA),immunoblot (e.g., Western), immunostrips (e.g., immunodipsticks)comprising a DLL4 binding protein of the invention adsorbed orimmobilized to substrate, FACS, and the like. Detection of DLL4 using aDLL4 binding protein of the invention may be conducted in vitro on amixture, solution, or in biological sample. A biological sample that maybe contacted with a binding protein of the invention to detect ormeasure DLL4 in the sample includes, but is not limited to, urine,saliva, oral swab (buccal, lingual, or throat swab), dermal swab, dermalscrape, rectal swab, vaginal swab, whole blood sample, plasma sample,serum sample, tissue biopsy, and any other sample obtained from anindividual by a procedure known in the art. In another embodiment, aDLL4 binding protein may be employed to detect DLL4 in vivo such asvarious tomography and scanning methods, including but not limited toX-ray computer assisted tomography (CT), magnetic resonance imaging(MRI), and positron emission tomography (PET).

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to DLL4 binding proteins, particularly anti-DLL4antibodies, or antigen-binding portions thereof that bind DLL4. An aminoacid sequence (SEQ ID NO:1) for human DLL4 is shown in Table 1 alongwith a corresponding DLL4 nucleotide coding sequence (SEQ ID NO:2).Various aspects of the invention relate to antibodies and antibodyfragments, and pharmaceutical compositions thereof, as well as nucleicacids, recombinant expression vectors, and host cells for making suchantibodies and fragments. Methods of using the antibodies of theinvention to detect human DLL4 or murine DLL4, methods to inhibit humanor mouse DLL4 and/or human or mouse VEGFR2 or VEGFR1 activity, either invitro or in vivo, and methods to regulate gene expression are alsoencompassed by the invention.

TABLE 1 Amino acid and nucleotide coding sequences for human DLL4.Sequence Sequence Kind of Sequence Identifier123456789012345678901234567890 Human DLL4 Amino SEQ ID NO: 1MAAASRSASGWALLLLVALWQQRAAGSGVF Acid SequenceQLQLQEFINERGVLASGRPCEPGCRTFFRV CLKHFQAVVSPGPCTFGTVSTPVLGTNSFAVRDDSSGGGRNPLQLPFNFTWPGTFSLIIE AWHAPGDDLRPEALPPDALISKIAIQGSLAVGQNWLLDEQTSTLTRLRYSYRVICSDNYY GDNCSRLCKKRNDHFGHYVCQPDGNLSCLPGWTGEYCQQPICLSGCHEQNGYCSKPAECL CRPGWQGRLCNECIPHNGCRHGTCSTPWQCTCDEGWGGLFCDQDLNYCTHHSPCKNGATC SNSGQRSYTCTCRPGYTGVDCELELSECDSNPCRNGGSCKDQEDGYHCLCPPGYYGLHCE HSTLSCADSPCFNGGSCRERNQGANYACECPPNFTGSNCEKKVDRCTSNPCANGGQCLNR GPSRMCRCRPGFTGTYCELHVSDCARNPCAHGGTCHDLENGLMCTCPAGFSGRRCEVRTS IDACASSPCFNRATCYTDLSTDTFVCNCPYGFVGSRCEFPVGLPPSFPWVAVSLGVGLAV LLVLLGMVAVAVRQLRLRRPDDGSREAMNNLSDFQKDNLIPAAQLKNTNQKKELEVDCGL DKSNCGKQQNHTLDYNLAPGPLGRGTMPGKFPHSDKLSGEKAPLRLHSEKPECRISAICS PRDSMYQSVCLISEERNECVIATEV Human DLL4SEQ ID NO: 2 atggcggcagcgtcccggagcgcctctggc Nucleotide Codingtgggcgctactgctgctggtggcactttgg Sequence cagcagcgcgcggccggctccggcgtcttccagctgcagctgcaggagttcatcaacgag cgcggcgtactggccagtgggcggccttgcgagcccggctgccggactttcttccgcgtc tgccttaagcacttccaggcggtcgtctcgcccggaccctgcaccttcgggaccgtctcc acgccggtattgggcaccaactccttcgctgtccgggacgacagtagcggcggggggcgc aaccctctccaactgcccttcaatttcacctggccgggtaccttctcgctcatcatcgaa gcttggcacgcgccaggagacgacctgcggccagaggccttgccaccagatgcactcatc agcaagatcgccatccagggctccctagctgtgggtcagaactggttattggatgagcaa accagcaccctcacaaggctgcgctactcttaccgggtcatctgcagtgacaactactat ggagacaactgctcccgcctgtgcaagaagcgcaatgaccacttcggccactatgtgtgc cagccagatggcaacttgtcctgcctgcccggttggactggggaatattgccaacagcct atctgtctttcgggctgtcatgaacagaatggctactgcagcaagccagcagagtgcctc tgccgcccaggctggcagggccggctgtgtaacgaatgcatcccccacaatggctgtcgc cacggcacctgcagcactccctggcaatgtacttgtgatgagggctggggaggcctgttt tgtgaccaagatctcaactactgcacccaccactccccatgcaagaatggggcaacgtgc tccaacagtgggcagcgaagctacacctgcacctgtcgcccaggctacactggtgtggac tgtgagctggagctcagcgagtgtgacagcaacccctgtcgcaatggaggcagctgtaag gaccaggaggatggctaccactgcctgtgtcctccgggctactatggcctgcattgtgaa cacagcaccttgagctgcgccgactccccctgcttcaatgggggctcctgccgggagcgc aaccagggggccaactatgcttgtgaatgtccccccaacttcaccggctccaactgcgag aagaaagtggacaggtgcaccagcaacccctgtgccaacgggggacagtgcctgaaccga ggtccaagccgcatgtgccgctgccgtcctggattcacgggcacctactgtgaactccac gtcagcgactgtgcccgtaacccttgcgcccacggtggcacttgccatgacctggagaat gggctcatgtgcacctgccctgccggcttctctggccgacgctgtgaggtgcggacatcc atcgatgcctgtgcctcgagtccctgcttcaacagggccacctgctacaccgacctctcc acagacacctttgtgtgcaactgcccttatggctttgtgggcagccgctgcgagttcccc gtgggcttgccgcccagcttcccctgggtggccgtctcgctgggtgtggggctggcagtg ctgctggtactgctgggcatggtggcagtggctgtgcggcagctgcggcttcgacggccg gacgacggcagcagggaagccatgaacaacttgtcggacttccagaaggacaacctgatt cctgccgcccagcttaaaaacacaaaccagaagaaggagctggaagtggactgtggcctg gacaagtccaactgtggcaaacagcaaaaccacacattggactataatctggccccaggg cccctggggcgggggaccatgccaggaaagtttccccacagtgacaagagcttaggagag aaggcgccactgcggttacacagtgaaaagccagagtgtcggatatcagcgatatgctcc cccagggactccatgtaccagtctgtgtgtttgatatcagaggagaggaatgaatgtgtc attgccacggaggtataa

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. The meaningand scope of the terms should be clear, however, in the event of anylatent ambiguity, definitions provided herein take precedent over anydictionary or extrinsic definition. Further, unless otherwise requiredby context, singular terms shall include pluralities and plural termsshall include the singular. In this application, the use of “or” means“and/or” unless stated otherwise. Furthermore, the use of the term“including,” as well as other forms, such as “includes” and “included,”is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise.

Generally, nomenclatures used in connection with, and techniques of,cell and tissue culture, molecular biology, immunology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Enzymatic reactions and purification techniques are performedaccording to manufacturer's specifications, as commonly accomplished inthe art or as described herein. The nomenclatures used in connectionwith, and the laboratory procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well known and commonly used in theart. Standard techniques are used for chemical syntheses, chemicalanalyses, pharmaceutical preparation, formulation, and delivery, andtreatment of patients.

That the present invention may be more readily understood, select termsare defined below.

The term “polypeptide” as used herein, refers to any polymeric chain ofamino acids. The terms “peptide” and “protein” are used interchangeablywith the term polypeptide and also refer to a polymeric chain of aminoacids. The term “polypeptide” encompasses native or artificial proteins,protein fragments and polypeptide analogs of a protein sequence. Apolypeptide may be monomeric or polymeric. Use of “polypeptide” hereinis intended to encompass polypeptide and fragments and variants(including fragments of variants) thereof, unless otherwise stated. Foran antigenic polypeptide, a fragment of polypeptide optionally containsat least one contiguous or nonlinear epitope of polypeptide. The preciseboundaries of the at least one epitope fragment can be confirmed usingordinary skill in the art. The fragment comprises at least about 5contiguous amino acids, such as at least about 10 contiguous aminoacids, at least about 15 contiguous amino acids, or at least about 20contiguous amino acids. A variant of polypeptide is as described herein.

The term “isolated protein” or “isolated polypeptide” is a protein orpolypeptide that by virtue of its origin or source of derivation is notassociated with naturally associated components that accompany it in itsnative state; is substantially free of other proteins from the samespecies; is expressed by a cell from a different species; or does notoccur in nature. Thus, a polypeptide that is chemically synthesized orsynthesized in a cellular system different from the cell from which itnaturally originates will be “isolated” from its naturally associatedcomponents. A protein may also be rendered substantially free ofnaturally associated components by isolation, using protein purificationtechniques well known in the art.

The term “recovering” as used herein, refers to the process of renderinga chemical species such as a polypeptide substantially free of naturallyassociated components by isolation, e.g., using protein purificationtechniques well known in the art.

The term “human DLL4” (abbreviated herein as “hDLL4” or “huDLL4”), asused herein, includes several EGF-like domains and a DSL domain that isrequired for receptor binding. The term includes a protein comprisingabout 74-75 kDa. The structure and deduced DNA and protein sequences ofhuman DLL4 is described further in, for example, Shutter et al., Genes &Dev., 4: 1313-1318 (2000). The term “human DLL4” is intended to includerecombinant human DLL4 (rh DLL4), which can be prepared by standardrecombinant expression methods.

“Biological activity”, as used herein with respect to DLL4, refers toall inherent biological properties of DLL4. Biological properties ofDLL4 include, but are not limited to, binding a Notch receptor,activating a Notch receptor, negatively regulating VEGF signaling,repressing VEGFR2, and inducing VEGR1.

The terms “specific binding” or “specifically binding”, as used herein,in reference to the interaction of an antibody, a protein, or a peptidewith a second chemical species, means that the interaction is dependentupon the presence of a particular structure (e.g., an antigenicdeterminant or epitope) on the chemical species; for example, anantibody recognizes and binds to a specific protein structure ratherthan to proteins generally. If an antibody is specific for epitope “A”,the presence of a molecule containing epitope A (or free, unlabeled A),in a reaction containing labeled “A” and the antibody, will reduce theamount of labeled A bound to the antibody.

A “binding protein” is a monomeric or multimeric protein that binds toand forms a complex with a binding partner, which may be a polypeptide,an antigen, a chemical compound or other molecule, or a substrate of anykind. A binding protein specifically binds a binding partner. Bindingproteins include antibodies and other molecules comprising one or moreantigen-binding domains that bind to an antigen molecule or a particularsite (epitope) on the antigen molecule. A binding protein includes anantibody or any of its antigen-binding fragments, and various forms andderivatives of antibodies known in the art and described below.Accordingly, a binding protein includes, but is not limited to, anantibody, a tetrameric immunoglobulin, an IgG molecule, an IgG₁molecule, a monoclonal antibody, a chimeric antibody, a CDR-graftedantibody, a humanized antibody, an affinity matured antibody, andfragments of any such antibodies that retain the ability to bind to anantigen.

The term “antibody”, as used herein, broadly refers to anyimmunoglobulin (Ig) molecule comprised of four polypeptide chains, twoheavy (H) chains and two light (L) chains, or any functional fragment,mutant, variant, or derivation thereof, which retains the essentialepitope binding features of an Ig molecule. Such mutant, variant, orderivative antibody formats are known in the art. Nonlimitingembodiments of which are discussed below.

In a full-length antibody, each heavy chain is comprised of a heavychain variable region (abbreviated herein as HCVR or VH) and a heavychain constant region. The heavy chain constant region is comprised ofthree domains, CH1, CH2 and CH3. Each light chain is comprised of alight chain variable region (abbreviated herein as LCVR or VL) and alight chain constant region. The light chain constant region iscomprised of one domain, CL. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed complementaritydetermining regions (CDR), interspersed with regions that are moreconserved, termed framework regions (FR). Each VH and VL is composed ofthree CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, and FR4. Immunoglobulin molecules can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1and IgA2) or subclass.

The term “Fc region” is used to define the C-terminal region of animmunoglobulin heavy chain, which may be generated by papain digestionof an intact antibody. The Fc region may be a native sequence Fc regionor a variant Fc region. The Fc region of an immunoglobulin generallycomprises two constant domains, a CH2 domain and a CH3 domain, andoptionally comprises a CH4 domain. Replacements of amino acid residuesin the Fc portion to alter antibody effector function are known in theart (U.S. Pat. Nos. 5,648,260 and 5,624,821). The Fc portion of anantibody mediates several important effector functions, e.g., cytokineinduction, ADCC, phagocytosis, complement dependent cytotoxicity (CDC),and half-life/clearance rate of antibody and antigen-antibody complexes.In some cases these effector functions are desirable for a therapeuticantibody but in other cases might be unnecessary or even deleterious,depending on the therapeutic objectives. Certain human IgG isotypes,particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRsand complement C1q, respectively. Neonatal Fc receptors (FcRn) are thecritical components determining the circulating half-life of antibodies.In still another embodiment, at least one amino acid residue is replacedin the constant region of the antibody, for example, the Fc region ofthe antibody, such that effector functions of the antibody are altered.The dimerization of two identical heavy chains of an immunoglobulin ismediated by the dimerization of CH3 domains and is stabilized by thedisulfide bonds within the hinge region (Huber et al., Nature, 264:415-420 (1976); Thies et al., J. Mol. Biol., 293: 67-79 (1999)).Mutation of cysteine residues within the hinge regions to prevent heavychain-heavy chain disulfide bonds will destabilize dimerization of CH3domains. Residues responsible for CH3 dimerization have been identified(Dall'Acqua, Biochem., 37: 9266-9273 (1998)). Therefore, it is possibleto generate a monovalent half-Ig. Interestingly, these monovalent halfIg molecules have been found in nature for both IgG and IgA subclasses(Seligman, Ann. Immunol., 129: 855-70 (1978); Biewenga et al., Clin.Exp. Immunol., 51: 395-400 (1983)). The stoichiometry of FcRn: Ig Fcregion has been determined to be 2:1 (West et al., Biochem., 39:9698-9708 (2000)), and half Fc is sufficient for mediating FcRn binding(Kim et al., Eur. J. Immunol., 24: 542-548 (1994)). Mutations to disruptthe dimerization of CH3 domain may not have greater adverse effect onits FcRn binding as the residues important for CH3 dimerization arelocated on the inner interface of CH3 b sheet structure, whereas theregion responsible for FcRn binding is located on the outside interfaceof CH2-CH3 domains. However, the half Ig molecule may have certainadvantages in tissue penetration due to its smaller size in comparisonto that of a regular antibody. In one embodiment, at least one aminoacid residue is replaced in the constant region of a binding protein ofthe invention, for example the Fc region, such that the dimerization ofthe heavy chains is disrupted, resulting in half Ig molecules. Theanti-inflammatory activity of IgG is completely dependent on sialylationof the N-linked glycan of the IgG Fc fragment. The precise glycanrequirements for anti-inflammatory activity has been determined, suchthat an appropriate IgG1 Fc fragment can be created, thereby generatinga fully recombinant, sialylated IgG1 Fc with greatly enhanced potency(Anthony et al., Science, 320: 373-376 (2008)).

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retains the ability to bind specifically to an antigen(i.e., to a particular epitope of an antigen, such as an epitope ofDLL4). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody. Suchantibody embodiments may also be bispecific, dual specific, ormulti-specific formats; specifically binding to two or more differentantigens (or two or more different epitopes of the same antigen).Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii)a F(ab′)₂ fragment, a bivalent fragment comprising two Fab fragmentslinked by a disulfide bridge at the hinge region; (iii) an Fd fragmentconsisting of the VH and CH1 domains; (iv) an Fv fragment consisting ofthe VL and VH domains of a single arm of an antibody; (v) a dAb fragment(Ward et al., Nature, 341:544-546 (1989); PCT Publication No. WO90/05144 A1), which comprises a single variable domain; and (vi) anisolated complementarity determining region (CDR). Furthermore, althoughthe two domains of the Fv fragment, VL and VH, are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair toform monovalent molecules (known as singlechain Fv (scFv); see, e.g., Bird et al., Science, 242: 423-426 (1988);Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988)). Suchsingle chain antibodies are also intended to be encompassed within theterm “antigen-binding portion” of an antibody. Other forms of singlechain antibodies, such as diabodies, are also encompassed. Diabodies arebivalent, bispecific antibodies in which VH and VL domains are expressedon a single polypeptide chain, but using a linker that is too short toallow for pairing between the two domains on the same chain, therebyforcing the domains to pair with complementary domains of another chainand creating two antigen binding sites (see, e.g., Holliger et al.,Proc. Natl. Acad. Sci. USA, 90: 6444-6448 (1993); Poljak, R. J.,Structure, 2: 1121-1123 (1994)). Such antibody binding portions areknown in the art (see, Kontermann and Dubel eds., Antibody Engineering(Springer-Verlag. New York, 2001), p. 790 (ISBN 3-540-41354-5)). Inaddition, single chain antibodies also include “linear antibodies”comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, togetherwith complementary light chain polypeptides, form a pair of antigenbinding regions (Zapata et al. Protein Eng., 8(10): 1057-1062 (1995);and U.S. Pat. No. 5,641,870).

The term “antibody construct” (or “DLL4 antibody construct”) as usedherein refers to a polypeptide comprising one or more the antigenbinding portions of the invention linked to a linker polypeptide or animmunoglobulin constant domain. Linker polypeptides comprise two or moreamino acid residues joined by peptide bonds and are used to link one ormore antigen binding portions. Such linker polypeptides are well knownin the art (see e.g., Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); Poljak, R. J., Structure, 2: 1121-1123 (1994)). Animmunoglobulin constant domain refers to a heavy or light chain constantdomain. Human IgG heavy chain and light chain constant domain amino acidsequences are known in the art and represented in Table 2.

TABLE 2 Sequence of human IgG heavy chain constant domain and light chain constant domain Sequence  Identi- Sequence Protein  fier 12345678901234567890123456789012 Ig SEQ IDASTKGPSVFFLAPSSKSTSGGTAALG gamma-1  NO: 3 CLVKDYFPEPVTVSWNSGALTSGVHTconstant FPAVLQSSGLYSLSSVVTVPSSSLGT region QTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSREEMTKNQVSLTCLVKGFYPSDIEAVEWESNGQPNNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGKIg SEQ ID ASTKGPSVFPLAPSSKSTSGGTAALG gamma-1  NO: 4CLVKDYFPEPVTVSWNSGALTSGVHT constant FPAVLQSSGLYSLSSVVTVPSSSLGT region QTYNICVNHKPSNTKVDKKVEPKSCD mutant KTHTCPPCPAPEAAGGPSVFLFPPKPLKDTMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALEKPAPITISKAKGQPREPQVYTLPPSNREEMTKQVSLTCLVKGFYPSDI AVE WESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HHEALNHYTQKSLSLSPGK Ig SEQ IDTVAAPSVFIFPPSDEQLKSGTASVVC Kappa NO: 5 LLNNFYPREAKVQWKVDNALQSGNSQconstant VESTEQDSKDSTYSLSSTLTLSKADY region EKHKVYACEVTHQGLSSPVTKSFNRG ECIg  SEQ ID QPKAAPSVTLFPPSSEELQANKATLV Lambda  NO: 6CLISDFYPGAVTVAWKADSSPVKAGV constant ETTTPSKQSNNKYAASSYLSLTPEQW regionKSHRSYSCQVTHEGSTVEKTVAPTECS

Still further, an antibody or antigen-binding portion thereof may bepart of a larger immunoadhesion molecule, formed by covalent ornoncovalent association of the antibody or antibody portion with one ormore other proteins or peptides. Examples of such immunoadhesionmolecules include use of the streptavidin core region to make atetrameric scFv molecule (Kipriyanov et al., Human Antibodies andHybridomas, 6: 93-101 (1995)) and use of a cysteine residue, a markerpeptide, and a C-terminal polyhistidine tag to make bivalent andbiotinylated scFv molecules (Kipriyanov et al., Mol. Immunol., 31:1047-1058 (1994)). Antibody portions, such as Fab and F(ab′)2 fragments,can be prepared from whole antibodies using conventional techniques,such as papain or pepsin digestion, respectively, of whole antibodies.Moreover, antibodies, antibody portions, and immunoadhesion moleculescan be obtained using standard recombinant DNA techniques, as describedherein and known in the art.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds hDLL4 is substantially free of antibodies that specifically bindantigens other than hDLL4). An isolated antibody that specifically bindshDLL4 may, however, have cross-reactivity to other antigens, such asDLL4 molecules from other species (e.g., muDLL4). Moreover, an isolatedantibody may be substantially free of other cellular material and/orchemicals.

The term “monoclonal antibody” and abbreviations “MAb” and “mAb”, asused herein, refers to an antibody obtained from a population ofsubstantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts. Monoclonalantibodies are highly specific, being directed against a single antigen.Furthermore, in contrast to polyclonal antibody preparations thattypically include different antibodies directed against differentdeterminants (epitopes), each mAb is directed against a singledeterminant on the antigen. The modifier “monoclonal” is not to beconstrued as requiring production of the antibody by any particularmethod.

The term “human antibody”, as used herein, is intended to includeantibodies having variable and constant regions derived from humangermline immunoglobulin sequences. The human antibodies of the inventionmay include amino acid residues not encoded by human germlineimmunoglobulin sequences (e.g., mutations introduced by random orsite-specific mutagenesis in vitro or by somatic mutation in vivo), forexample in the CDRs and in particular CDR3. However, the term “humanantibody”, as used herein, is not intended to include antibodies inwhich CDR sequences derived from the germline of another mammalianspecies, such as a mouse, have been grafted onto human frameworksequences.

The term “recombinant human antibody”, as used herein, is intended toinclude all human antibodies that are prepared, expressed, created, orisolated by recombinant means, such as antibodies expressed using arecombinant expression vector transfected into a host cell, antibodiesisolated from a recombinant, combinatorial human antibody library(Hoogenboom, Trends Biotechnol., 15:62-70 (1997); Azzazy and Highsmith,Clin. Biochem., 35: 425-445 (2002); Gavilondo and Larrick,BioTechniques, 29: 128-145 (2000); Hoogenboom and Chames, Immunol.Today, 21: 371-378 (2000)), antibodies isolated from an animal (e.g., amouse) that is transgenic for human immunoglobulin genes (see, Taylor etal., Nucl. Acids Res., 20: 6287-6295 (1992); Kellermann and Green, Curr.Opin. Biotechnol., 13: 593-597 (2002); Little et al., Immunol. Today,21: 364-370 (2000)) or antibodies prepared, expressed, created orisolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinanthuman antibodies have variable and constant regions derived from humangermline immunoglobulin sequences. In certain embodiments, however, suchrecombinant human antibodies are subjected to in vitro mutagenesis (or,when an animal transgenic for human Ig sequences is used, in vivosomatic mutagenesis) and thus the amino acid sequences of the VH and VLregions of the recombinant antibodies are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human antibody germline repertoire in vivo.

The term “chimeric antibody” refers to antibodies which comprise heavyand light chain variable region sequences from one species and constantregion sequences from another species, such as antibodies having murineheavy and light chain variable regions linked to human constant regions.

As used herein, the term “CDR” refers to a complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and the light chain, whichare designated “CDR1”, “CDR2”, and “CDR3”, for each of the variableregions. The term “CDR set” as used herein refers to a group of threeCDRs that occur in a single variable region that binds the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as “Kabat CDRs”. Chothiaand coworkers (Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987);Chothia et al., Nature, 342: 877-883 (1989)) found that certainsub-portions within Kabat CDRs adopt nearly identical peptide backboneconformations, despite having great diversity at the level of amino acidsequence. These sub-portions were designated as “L1”, “L2”, and “L3”, or“H1”, “H2”, and “H3”, where the “L” and the “H” designate the lightchain and the heavy chain regions, respectively. These regions may bereferred to as “Chothia CDRs”, which have boundaries that overlap withKabat CDRs. Other boundaries defining CDRs overlapping with the KabatCDRs have been described by Padlan, FASEB J., 9: 133-139 (1995) andMacCallum, J. Mol. Biol., 262(5): 732-745 (1996). Still other CDRboundary definitions may not strictly follow one of the herein systems,but will nonetheless overlap with the Kabat CDRs, although they may beshortened or lengthened in light of prediction or experimental findingsthat particular residues or groups of residues or even entire CDRs donot significantly impact antigen binding. The methods used herein mayutilize CDRs defined according to any of these systems, although certainembodiments use Kabat or Chothia defined CDRs.

The terms “Kabat numbering,” “Kabat definitions”, and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e., hypervariable) than other amino acid residues inthe heavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al., Ann. NY Acad. Sci., 190: 382-391(1971) and Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242 (1991)). For the heavy chain variable region(VH), the hypervariable region ranges from amino acid positions 31 to 35for CDR1, amino acid positions 50 to 65 for CDR2, and amino acidpositions 95 to 102 for CDR3. For the light chain variable region (VL),the hypervariable region ranges from amino acid positions 24 to 34 forCDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions89 to 97 for CDR3.

The growth and analysis of extensive public databases of amino acidsequences of variable heavy and light regions over the past twenty yearshave led to the understanding of the typical boundaries betweenframework regions (FR) and CDR sequences within variable regionsequences and enabled persons skilled in this art to accuratelydetermine the CDRs according to Kabat numbering, Chothia numbering, orother systems. See, e.g., Martin, “Protein Sequence and StructureAnalysis of Antibody Variable Domains,” In Kontermann and Dübel, eds.,Antibody Engineering (Springer-Verlag, Berlin, 2001), chapter 31, pages432-433. A useful method of determining the amino acid sequences ofKabat CDRs, and thereby sequences of Kabat FRs as well, within the aminoacid sequences of variable heavy (VH) and variable light (VL) regions isprovided below:

To identify a CDR-L1 amino acid sequence:

-   -   Starts approximately 24 amino acid residues from the amino        terminus of the VL region;    -   Residue before the CDR-L1 sequence is always cysteine (C);    -   Residue after the CDR-L1 sequence is always tryptophan (W),        typically Trp-Tyr-Gln (W-Y-Q), but also Trp-Leu-Gln (W-L-Q),        Trp-Phe-Gln (W-F-Q), and Trp-Tyr-Leu (W-Y-L);    -   Length is typically 10 to 17 amino acid residues.

To identify a CDR-L2 amino acid sequence:

-   -   Starts always 16 residues after the end of CDR-L1;    -   Residues before the CDR-L2 sequence are generally Ile-Tyr (I-Y),        but also Val-Tyr (V-Y), Ile-Lys (I-K), and Ile-Phe (I-F);    -   Length is always 7 amino acid residues.

To identify a CDR-L3 amino acid sequence:

-   -   Starts always 33 amino acids after the end of CDR-L2;    -   Residue before the CDR-L3 amino acid sequence is always a        cysteine (C);    -   Residues after are always Phe-Gly-X-Gly (F-G-X-G) (SEQ ID NO:7),        where X is any amino acid;    -   Length is typically 7 to 11 amino acid residues.

To identify a CDR-H1 amino acid sequence:

-   -   Starts approximately 31 amino acid residues from amino terminus        of VH region and always 9 residues after a cysteine (C);    -   Residues before are always Cys-X-X-X-X-X-X-X-X (SEQ ID NO:8),        where X is any amino acid;    -   Residue after is always a Trp (W), typically Trp-Val (W-V), but        also Trp-Ile (W-I), and Trp-Ala (W-A);    -   Length is typically 5 to 7 amino acid residues.

To identify a CDR-H2 amino acid sequence:

-   -   Starts always 15 amino acid residues after the end of CDR-H1;    -   Residues before are typically Leu-Glu-Trp-Ile-Gly (L-E-W-I-G)        (SEQ ID NO:9), but other variations also;    -   Residues after are        Lys/Arg-Leu/Ile/Val/Phe/Thr/Ala-Thr/Ser/Ile/Ala        (K/R-L/I/V/F/T/A-T/S/I/A);    -   Length is typically 16 to 19 amino acid residues.

To identify a CDR-H3 amino acid sequence:

-   -   Starts always 33 amino acid residues after the end of CDR-H2 and        always 3 after a cysteine (C)    -   Residues before are always Cys-X-X (C-X-X), where X is any amino        acid, typically Cys-Ala-Arg (C-A-R);    -   Residues after are always Trp-Gly-X-Gly (W-G-X-G) (SEQ ID        NO:10), where X is any amino acid;    -   Length is typically 3 to 25 amino acid residues.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which comprise heavyand light chain variable region sequences from a non-human species(e.g., a mouse) but in which at least a portion of the VH and/or VLsequence has been altered to be more “human-like,” i.e., more similar tohuman germline variable sequences. A “humanized antibody” is an antibodyor a variant, derivative, analog, or fragment thereof, whichimmunospecifically binds to an antigen of interest and which comprises aframework (FR) region having substantially the amino acid sequence of ahuman antibody and a complementary determining region (CDR) havingsubstantially the amino acid sequence of a non-human antibody. As usedherein, the term “substantially” in the context of a CDR refers to a CDRhaving an amino acid sequence at least 80%, at least 85%, at least 90%,at least 95%, at least 98% or at least 99% identical to the amino acidsequence of a non-human antibody CDR. A humanized antibody comprisessubstantially all of at least one, and typically two, variable domains(Fab, Fab′, F(ab′) 2, FabC, Fv) in which all or substantially all of theCDR regions correspond to those of a non-human immunoglobulin (i.e.,donor antibody) and all or substantially all of the framework regionsare those of a human immunoglobulin consensus sequence. In anembodiment, a humanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain.

A humanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA, and IgE, and any isotype, includingwithout limitation IgG1, IgG2, IgG3, and IgG4. A humanized antibody maycomprise sequences from more than one class or isotype, and particularconstant domains may be selected to optimize desired effector functionsusing techniques well known in the art.

The framework regions and CDRs of a humanized antibody need notcorrespond precisely to the parental sequences, e.g., the donor antibodyCDR or the consensus framework may be mutagenized by substitution,insertion, and/or deletion of at least one amino acid residue so thatthe CDR or framework residue at that site does not correspond to eitherthe donor antibody or the consensus framework. In a preferredembodiment, such mutations, however, will not be extensive. Usually, atleast 80%, preferably at least 85%, more preferably at least 90%, andmost preferably at least 95% of the humanized antibody residues willcorrespond to those of the parental FR and CDR sequences. As usedherein, the term “consensus framework” refers to the framework region inthe consensus immunoglobulin sequence. As used herein, the term“consensus immunoglobulin sequence” refers to the sequence formed fromthe most frequently occurring amino acids (or nucleotides) in a familyof related immunoglobulin sequences (See, e.g., Winnaker, From Genes toClones (Verlagsgesellschaft, Weinheim, 1987)). A “consensusimmunoglobulin sequence” may thus comprise a “consensus frameworkregion(s)” and/or a “consensus CDR(s)”. In a family of immunoglobulins,each position in the consensus sequence is occupied by the amino acidoccurring most frequently at that position in the family. If two aminoacids occur equally frequently, either can be included in the consensussequence.

An “affinity matured” antibody is an antibody with one or morealterations in one or more CDRs thereof which result in an improvementin the affinity of the antibody for a target antigen, compared to aparent antibody which does not possess the alteration(s). Exemplaryaffinity matured antibodies will have nanomolar or even picomolaraffinities for the target antigen. A variety of procedures for producingaffinity matured antibodies are known in the art. For example, Marks etal., BioTechnology, 10: 779-783 (1992) describes affinity maturation byVH and VL domain shuffling. Random mutagenesis of CDR and/or frameworkresidues is described by Barbas et al., Proc. Nat. Acad. Sci. USA, 91:3809-3813 (1994); Schier et al., Gene, 169: 147-155 (1995); Yelton etal., J. Immunol., 155: 1994-2004 (1995); Jackson et al., J. Immunol.,154(7): 3310-3319 (1995); Hawkins et al, J. Mol. Biol., 226: 889-896(1992). Selective mutation at selective mutagenesis positions and atcontact or hypermutation positions with an activity enhancing amino acidresidue is described in U.S. Pat. No. 6,914,128 B1.

The term “multivalent binding protein” denotes a binding proteincomprising two or more antigen binding sites (also referred to herein as“antigen binding domains”). A multivalent binding protein is preferablyengineered to have three or more antigen binding sites, and is generallynot a naturally occurring antibody. The term “multispecific bindingprotein” refers to a binding protein capable of binding two or morerelated or unrelated targets, including a binding protein capable ofbinding two or more different epitopes of the same target molecule.

The term “bispecific antibody”, as used herein, refers tofull-lengthantibodies that are generated by quadroma technology (see Milstein etal., Nature, 305(5934): 537-540 (1983)), by chemical conjugation of twodifferent monoclonal antibodies (see, Staerz et al., Nature, 314(6012):628-631 (1985)), or by knob-into-hole or similar approaches whichintroduces mutations in the Fc region (see Holliger et al., Proc. Natl.Acad. Sci. USA, 90(14): 6444-6448 (1993)), resulting in multipledifferent immunoglobulin species of which only one is the functionalbispecific antibody. By molecular function, a bispecific antibody bindsone antigen (or epitope) on one of its two binding arms (one pair ofHC/LC), and binds a different antigen (or epitope) on its second arm (adifferent pair of HC/LC). By this definition, a bispecific antibody hastwo distinct antigen binding arms (in both specificity and CDRsequences), and is monovalent for each antigen it binds to.

The term “dual-specific antibody”, as used herein, refers tofull-lengthantibodies that can bind two different antigens (or epitopes) in each ofits two binding arms (a pair of HC/LC) (see PCT publication WO02/02773). Accordingly a dual-specific binding protein has two identicalantigen binding arms, with identical specificity and identical CDRsequences, and is bivalent for each antigen to which it binds.

“Dual variable domain” (“DVD”) binding proteins of the inventioncomprise two or more antigen binding sites and may be divalent (twoantigen binding sites), tetravalent (four antigen binding sites), ormultivalent binding proteins. DVDs may be monospecific, i.e., capable ofbinding one antigen (or one specific epitope), or multispecific, i.e.,capable of binding two or more antigens (i.e., two or more epitopes ofthe same target antigen molecule or two or more epitopes of differenttarget antigens). A preferred DVD binding protein comprises two heavychain DVD polypeptides and two light chain DVD polypeptides is referredto as a “DVD immunoglobulin” or “DVD-Ig”. Such a DVD-Ig binding proteinis thus tetrameric and reminiscent of an IgG molecule, but provides moreantigen binding site than an IgG molecule. Thus, each half of atetrameric DVD-Ig molecule is reminiscent of one half of an IgG moleculeand comprises a heavy chain DVD polypeptide and a light chain DVDpolypeptide, but unlike a pair of heavy and light chains of an IgGmolecule that provide a single antigen bindind domain, a pair of heavyand light chains of a DVD-Ig provide two or more antigen binding sites.

Each antigen binding site of a DVD-Ig binding protein is derived from adonor (“parental”) monoclonal antibody and thus comprises a heavy chainvariable domain (VH) and a light chain variable domain (VL) with a totalof six CDRs involved in antigen binding per antigen binding site.Accordingly, a DVD-Ig binding protein that binds two different epitopes(i.e., two different epitopes of two different antigen molecules or twodifferent epitopes of the same antigen molecule) comprises an antigenbinding site derived from a first parental monoclonal antibody and anantigen binding site of a second parental monoclonal antibody.

A description of the design, expression, and characterization of DVD-Igbinding molecules is provided in PCT Publication No. WO 2007/024715,U.S. Pat. No. 7,612,181, and Wu et al., Nature Biotech., 25: 1290-1297(2007). A preferred example of such DVD-Ig molecules comprises a heavychain that comprises the structural formula VD1-(X1)n-VD2-C-(X2)n,wherein VD1 is a first heavy chain variable domain, VD2 is a secondheavy chain variable domain, C is a heavy chain constant domain, X1 is alinker with the proviso that it is not CH1, X2 is an Fc region, and n is0 or 1, but preferably 1; and a light chain that comprises thestructural formula VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first lightchain variable domain, VD2 is a second light chain variable domain, C isa light chain constant domain, X1 is a linker with the proviso that itis not CH1, and X2 does not comprise an Fc region; and n is 0 or 1, butpreferably 1. Such a DVD-Ig may comprise two such heavy chains and twosuch light chains, wherein each chain comprises variable domains linkedin tandem without an intervening constant region between variableregions, wherein a heavy chain and a light chain associate toform tandemfunctional antigen binding sites, and a pair of heavy and light chainsmay associate with another pair of heavy and light chains toform atetrameric binding protein with four functional antigen binding sites.In another example, a DVD-Ig molecule may comprise heavy and lightchains that each comprise three variable domains (VD1, VD2, VD3) linkedin tandem without an intervening constant region between variabledomains, wherein a pair of heavy and light chains may associate toformthree antigen binding sites, and wherein a pair of heavy and lightchains may associate with another pair of heavy and light chains toforma tetrameric binding protein with six antigen binding sites.

In a preferred embodiment, a DVD-Ig binding protein according to theinvention not only binds the same target molecules bound by its parentalmonoclonal antibodies, but also possesses one or more desirableproperties of one or more of its parental monoclonal antibodies.Preferably, such an additional property is an antibody parameter of oneor more of the parental monoclonal antibodies. Antibody parameters thatmay be contributed to a DVD-Ig binding protein from one or more of itsparental monoclonal antibodies include, but are not limited to, antigenspecificity, antigen affinity, potency, biological function, epitoperecognition, protein stability, protein solubility, productionefficiency, immunogenicity, pharmacokinetics, bioavailability, tissuecross reactivity, and orthologous antigen binding.

A DVD-Ig binding protein according to the invention binds at least oneepitope of a human DLL4 protein. Non-limiting examples of a DVD-Igbinding protein according to the invention include a DVD-Ig bindingprotein that binds one or more epitopes of human DLL4, a DVD-Ig bindingprotein that binds an epitope of a human DLL4 and an epitope of a DLL4of another species (for example, mouse), and a DVD-Ig binding proteinthat binds an epitope of a human DLL4 and an epitope of another targetmolecule (for example, VEGFR2 or VEGFR1).

A “functional antigen binding site” of a binding protein is one that iscapable of binding a target antigen. The antigen binding affinity of theantigen binding site is not necessarily as strong as the parent antibodyfrom which the antigen binding site is derived, but the ability to bindantigen must be measurable using any one of a variety of methods knownfor evaluating antibody binding to an antigen. Moreover, the antigenbinding affinity of each of the antigen binding sites of a multivalentantibody herein need not be quantitatively the same.

As used herein, the terms “acceptor” and “acceptor antibody” refer to anantibody or nucleic acid sequence providing or encoding at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions (FRs). Insome embodiments, the term “acceptor” refers to the antibody amino acidor nucleic acid sequence providing or encoding the constant region(s).In yet another embodiment, the term “acceptor” refers to the antibodyamino acid or nucleic acid sequence providing or encoding one or more ofthe framework regions and the constant region(s). In a specificembodiment, the term “acceptor” refers to a human antibody amino acid ornucleic acid sequence that provides or encodes at least 80%, preferably,at least 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. Inaccordance with this embodiment, an acceptor may contain at least 1, atleast 2, at least 3, least 4, at least 5, or at least 10 amino acidresidues that does (do) not occur at one or more specific positions of ahuman antibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell-known in the art, antibodies in development, or antibodiescommercially available).

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (J. Mol. Biol., 196: 901-917 (1987); Chothiaet al., J. Mol. Biol., 227: 799-817 (1992), both are incorporated hereinby reference). According to Chothia et al., critical portions of theCDRs of many antibodies have nearly identical peptide backboneconfirmations despite great diversity at the level of amino acidsequence. Each canonical structure specifies primarily a set of peptidebackbone torsion angles for a contiguous segment of amino acid residuesforming a loop.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In a preferred embodiment, thedonor antibody is an antibody from a species different from the antibodyfrom which the framework regions are obtained or derived. In the contextof a humanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined by differentsystems (for example, see above), the meaning of a framework sequence issubject to correspondingly different interpretations. The six CDRs(CDR-L1, -L2, and -L3 of light chain and CDR-H1, -H2, and -H3 of heavychain) also divide the framework regions on the light chain and theheavy chain intofour sub-regions (FR1, FR2, FR3, and FR4) on each chain,in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 andFR3, and CDR3 between FR3 and FR4. Without specifying the particularsub-regions as FR1, FR2, FR3, or FR4, a framework region, as referred byothers, represents the combined FRs within the variable region of asingle, naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region.

Human heavy chain and light chain framework (FR) sequences are known inthe art that can be used as heavy chain and light chain “acceptor”framework sequences (or simply, “acceptor” sequences) to humanize anon-human antibody using techniques known in the art. In an embodimentof the invention, human heavy chain and light chain acceptor sequencesare selected from the framework sequences listed in publicly availabledatabases such as V-base (hypertext transferprotocol://vbase.mrc-cpe.cam.ac.uk/) or in the internationalImMunoGeneTics® (IMGT®) information system (hypertext transferprotocol://imgt.cines.fr/texts/IMGTrepertoire/LocusGenes/). Table 3,below, provides a non-limiting list of examples of human heavy chainacceptor sequences known in the art. Table 4, below, provides anon-limiting list of examples of human light chain acceptor sequencesknown in the art. In an embodiment of the invention, human heavy chainand light chain acceptor sequences are selected from the amino acidsequences described in Table 3 and Table 4, below, however, other humanheavy chain and light acceptors sequences not listed in Tables 3 and 4may also be used to humanize an antibody according to the invention.

TABLE 3  Heavy Chain Acceptor Sequences. Protein region/ SEQ ClosestAmino Acid Sequence ID Germline  12345678901234567 NO: Family890123456789012 11 VH3-7 FR1 EVQLVESGGGLVQPGG  SLRLSCAASGFTFS 12VH3-7 FR2 WVRQAPGKGLEWVA 13 VH3-7 FR3 RFTISRDNAKNSLYL  QMNSLRAEDTAVYYCAR14 JH4 FR4 WGQGTLVTVSS 15 VH3 CONSENUSUS EVQLVESGGGLVQPGG  FR1SLRLSCAASGFTFS 16 VH3 CONSENUSUS WVRQAPGKGLEWVS FR2 17 VH3 CONSENUSUSRFTISRDNSKNTLYLQM  FR3 NSLRAEDTAVYYCAR 18 JH4 FR4 WGQGTLVTVSS 19VH1-46 FR1 QVQLVQSGAEVKKPGA  SVKVSCKASGYTFT 20 VH1-46FR2 WVRQAPGQGLEWMG21 VH1-46 FR3 RVTMTRDTSTSTVYM  ELSSLRSEDTAVYYCAR 22 JH4 FR4 WGQGTLVTVSS23 VH3-30 FR1 QVQLVESGGGVVQPG  RSLRLSCAASGFTFS 24 VH3-30 FR2WVRQAPGKGLEWVA 25 VH3-30 FR3 RFTISRDNSKNTLYLQM NSLRAEDTAVYYCAR 26JH3 FR4 WGQGTMVTVSS 27 VH3 CONSENUSUS EVQLVESGGGLVQPGGS FR1LRLSCAASGFTFS 28 VH3 CONSENUSUS WVRQAPGKGLEWVS FR2 29 VH3 CONSENUSUSRFTISRDNSKNTLYLQ FR3 MNSLRAEDTAVYYCAR 30 JH3 FR4 WGQGTMVTVSS 31VH2-70/JH6 FR1 EVTLRESGPALVKPT QTLTLTCTFSGFSLS 32 VH2-70/JH6 FR2WIRQPPGKALEWLA 33 VH2-70/JH6 FR3 RLT1SKDTSKNQVVLT MTNMDPVDTATYYCAR 34VH2-70/JH6 FR4 WGQGTTVTVSS 35 VH2-26/JH6 FR1 EVTLKESGPVLVKPTETLTLTCTVSGFSLS 36 VH2-26/JH6 FR2 WIRQPPGKALEWLA 37 VH2-26/JH6 RLTISKDTSKSQVVLT FR3 MTNMDPVDTATYYCAR 38 VH2-26/JH6 FR4 WGQGTTVTVSS 39VH3-72/JH6 FR1 EVQLVESGGGLVQPGG SLRLSCAASGFTFS 40 VH3-72/JH6 FR2WVRQAPGKGLEWVG

TABLE 4 Light Chain Acceptor Sequences Protein region/ SEQ Closest IDGermline Sequence NO.: Family 12345678901234567890123456789012  83O2 FR1 DIQMTQSPSSLSASVGDRVTITC  84 O2 FR2 WYQQKPGKAPKLLIY  85 O2 FR3GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC  86 JK2 FR4 FGQGTKLEIK  87 L2 FR1EIVMTQSPATLSVSPGERATLSC  88 L2 FR2 WYQQKPGQAPRLLIY  89 L2 FR3GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC  90 JK2 FR4 FGQGTKLEIK  91 B3/JK4 FR1DIVMTQSPDSLAVSLGERATINC  92 B3/JK4 FR2 WYQQKPGQPPKLLIY  93 B3/JK4 FR3GVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC  94 B3/JK4 FR4 FGGGTKVEIKR  95L2/JK4 FR1 EIVMTQSPATLSVSPGERATLSC  96 L2/JK4 FR2 WYQQKPGQAPRLLIY  97L2/JK4 FR3 GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC  98 L2/JK4 FR4 FGGGTKVEIKR 99 L15/JK4 FR1 DIQMTQSPSSLSASVGDRVTITC 100 L15/JK4 FR2 WYQQKPEKAPKSLIY101 L15/JK4 FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 102 L15/JK4 FR4FGGGTKVEIKR 103 L5/JK4 FR1 DIQMTQSPSSVSASVGDRVTITC 104 L5/JK4 FR2WYQQKPGKAPKLLIY 105 L5/JK4 FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC 106L5/JK4 FR4 FGGGTKVEIKR 107 IGLV3-1 FR1 SYELTQPPSVSVSPGQTASITC 108IGLV3-1 FR2 WYQQKPGQSPVLVIY 109 IGLV3-1 FR3GIPERFSGSNSGDTATLTISGTQPMDEADYYC 110 IGLV3-1/Th  FGYGTKVTVL FR4 111IGLV3-1 FR1 SYELTQPPSVSVSPGQTASITC 112 IGLV3-1 FR2 WYQQKPGQSPVLVIY 113IGLV3-1 FR3 GIPERFSGSNSGDTATLTISGTQPMDEADYYC 114 IGLV3-1/Th  GGGTKLTVLGFR4 115 IGLV3-1 FR1 YELTQPPSVSVSPGQTASITC 116 IGLV3-1 FR2WYQQKPGQSPVLVIY 117 IGLV3-1 FR3 GIPERFSGSNSGDTATLTISGTQPMDEADYYC 118IGLV3-1/ GGGTKLTVLG Th FR4 119 IGLV3-1 FR1 LYVLTQPPSVSVSPGQTASITC 120IGLV3-1 FR2 WYQQKPGQSPVLVIY 121 IGLV3-1 FR3GIPERFSGSNSGDTATLTISGTQTMDEADYLC 122 IGLV3-1/Th  FGGGTKVTVLG FR4 123IGKV6D-21 EYVLTQSPDFQSVTPKEKVTITC FR1 124 IGKV6D-21 WYQQKPDQSPKLVIY FR2125 IGKV6D-21 GVPSRFSGSNSGDDATLTINSLEAEDAATYYC FR3 126 IGKV6D-21/FGQGTKVEIKR JK FR4 127 IGKV3D-15 EYVLTQSPATLSVSPGERATLSC FR1 128IGKV3D-15 WYQQKPGQSPRLVIY FR2 129 IGKV3D-15DIPARFSGSNSGDEATLTISSLQSEDFAVYYC FR3 130 IGKV3D-15/ FGQGTRLEIKR JK FR4131 IGKV4-1 FR1 DYVLTQSPDSLAVSLGERATINC 132 IGKV4-1 FR2 WYQQKPGQSPKLVIY133 IGKV4-1 FR3 GIPDRFSGSNSGDDATLTISSLQAEDVAVYYC 134 IGKV4-1/JK FGGGTKVEIKR FR4 135 IGLV3-1 FR1 LPVLTQPPSVSVSPGQTASITC 136 IGLV3-1 FR2WYQQKPGQSPVLVIY 137 IGLV3-1 FR3 GIPERFSGSNSGNTATLTISGTQTMDEADYLC 138IGLV3-1/Th  FGGGTKVTVL FR4 139 IGLV3-1 FR1 SYELTQPPSVSVSPGQTASITC 140IGLV3-1 FR2 WYQQKPGQSPVLVIY 141 IGLV3-1 FR3GIPERFSGSNSGNTATLTISGTQTMDEADYLC 142 IGLV3-1/Th  FGGGTKLTVL FR4

In an embodiment, heavy chain human acceptor framework sequences fromTable 3 for use in generating humanized antibodies that bind DLL4according to the invention include a set consisting of the VH3-7 FR1,the VH3-7 FR2, the VH3-7 FR3, and the JH4 FR4 acceptor sequences; a setconsisting of the VH3 consensus FR1, the VH3 consensus FR2, the VH3consensus FR3, and the JH4 FR4 acceptor sequences; a set consisting ofthe VH1-46 FR1, the VH1-46 FR2, the VH1-46 FR3, and the JH4 FR4 acceptorsequences; a set consisting of the VH3-30 FR1, the VH3-30 FR2, theVH3-30 FR3, and the JH3 FR4 acceptor sequences; and a set consisting ofthe VH3 consensus FR1, the VH3 consensus FR2, the VH3 consensus FR3, andthe JH3 FR4 acceptor sequences.

In an embodiment, light chain human acceptor framework sequences fromTable 4 for use in generating humanized antibodies that bind DLL4according to the invention include a set consisting of the O2 FR1, O2FR2, O2 FR3, and JK2 FR4 acceptors sequences and a set a consisting ofthe L2 FR1, L2 FR2, L2 FR3, and JK2 FR4 acceptor sequences.

In an embodiment, a set of human acceptor framework sequences for use ingenerating a humanized antibody that binds DLL4 according to theinvention comprises one or more (e.g., any one, two, three, four, five,six, seven, or eight per binding domain) of the acceptor frameworksequences selected from the group consisting of:

-   -   heavy chain framework-1 (H-FR1):    -   E-V-Q-L-V-E-S-G-G-G-L-V-Q-P-G-G-S-L-R-L-S-C-A-A-S-G-F-T-F-X₃₀        (SEQ ID NO:143), wherein X₃₀ is S, R, or G;    -   heavy chain framework-2 (H-FR2): W-V-R-Q-A-P-G-K-G-L-E-W-V-A        (SEQ ID NO:144);    -   heavy chain framework-3 (H-FR3):    -   R-F-T-I-S-R-D-N-A-K-X₁₁-S-L-Y-L-Q-M-N-S-L-R-A-E-D-T-A-V-Y-Y-C-X₃₁-R        (SEQ ID NO:145), wherein;    -   X₁₁ is N or S; and    -   X₃₁ is A or S;    -   heavy chain framework-4 (H-FR4): W-G-Q-G-T-L-V-T-V—S-S(SEQ ID        NO:146);    -   light chain framework-1 (L-FR1):    -   D-I-Q-M-T-Q-S—P-S-S-L-S-A-S-V-G-D-R-V-T-I-T-C (SEQ ID NO:147);    -   light chain framework-2 (L-FR2):        W-Y-Q-Q-K-P-G-K-X₉-P-K-L-L-I-X₁₅ (SEQ ID NO:148), wherein;    -   X₉ is A or S; and    -   X₁₅ is F or Y;    -   light chain framework-3 (L-FR3):    -   G-V-P-S-R-F-S-G-S-G-S-G-T-D-X₁₅-T-L-T-I-S-S-L-Q-P-E-D-F-A-T-Y-Y-C        (SEQ ID NO:149), wherein;    -   X₁₅ is F or S; and    -   light chain framework-4 (L-FR4): F-G-Q-G-T-K-L-E-I-K (SEQ ID        NO:150).

In a preferred embodiment, an antibody that binds DLL4 according to theinvention is humanized using a set of human acceptor sequencesconsisting of an H-FR1, H-FR2, H-FR3, H-FR-4, L-FR1, L-FR2, L-FR3, andL-FR4 acceptor sequence described above.

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol., 22(3):183-200 (2002); Marchalonis et al., Adv. Exp. Med. Biol., 484:13-30(2001)). One of the advantages provided by various embodiments of thepresent invention stems from the recognition that germline antibodygenes are more likely than mature antibody genes to conserve essentialamino acid sequence structures characteristic of individuals in thespecies, hence less likely to be recognized as from a foreign sourcewhen used therapeutically in that species.

As used herein, the term “key residue” refers to certain residues withinthe variable region that have more impact on the binding specificityand/or affinity of an antibody, in particular a humanized antibody. Akey residue includes, but is not limited to, one or more of thefollowing: a residue that is adjacent to a CDR, a potentialglycosylation site (can be either N- or O-glycosylation site), a rareresidue, a residue capable of interacting with the antigen, a residuecapable of interacting with a CDR, a canonical residue, a contactresidue between heavy chain variable region and light chain variableregion, a residue within the Vernier zone, and a residue in the regionthat overlaps between the Chothia definition of a variable heavy chainCDR1 and the Kabat definition of the first heavy chain framework.

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter (J. Mol. Biol., 224: 487-499 (1992)).Vernier zone residues form a layer underlying the CDRs and may impact onthe structure of CDRs and the affinity of the antibody.

As used herein, the term “neutralizing” refers to counteracting thebiological activity of an antigen when a binding protein specificallybinds the antigen. In an embodiment, the neutralizing binding proteinbinds an antigen and reduces its biological activity by at least about20%, 40%, 60%, 80%, 85%, 90%, 95%, or more.

The term “activity” includes activities such as the bindingspecificity/affinity of an antibody for an antigen, for example, ananti-hDLL4 antibody that binds to an DLL4 antigen and/or theneutralizing potency of an antibody, or an anti-hDLL4 antibody whosebinding to hDLL4 inhibits the biological activity of hDLL4, e.g.inhibition of PHA blast proliferation or inhibition of receptor bindingin a human Notch receptor binding assay, or PHA blast interferon-gammainduction assay.

The term “epitope” includes any polypeptide determinant thatspecifically binds to an immunoglobulin or T-cell receptor. In certainembodiments, epitope determinants include chemically active surfacegroupings of molecules such as amino acids, sugar side chains,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. An epitope is a region of an antigen that is bound byan antibody. An epitope thus consists of the amino acid residues of aregion of an antigen (or fragment thereof) known to bind to thecomplementary site on the specific binding partner. An antigen orantigenic fragment can contain more than one epitope. Thus, it isunderstood by persons skilled in this art that every “antigen bindingsite” of an antibody molecule binds an epitope of an antigen moleculeand every antigen molecule may have one, two, several, or many epitopes.Moreover, it is understood by persons skilled in this art that twoindependently isolated antibodies to an antigen molecule may bind at thesame epitope or at two different epitopes on the antigen molecule.

In certain embodiments, an antibody is said to specifically bind anantigen when it recognizes its target antigen in a complex mixture ofproteins and/or macromolecules. Antibodies are said to “bind to the sameepitope” if the antibodies cross-compete (one prevents the binding ormodulating effect of the other). In addition, structural definitions ofepitopes (overlapping, similar, identical) are informative, butfunctional definitions are often more relevant as they encompassstructural (binding) and functional (modulation, competition)parameters.

The term “surface plasmon resonance,” as used herein, refers to anoptical phenomenon that allows for the analysis of real-time biospecificinteractions by detection of alterations in protein concentrationswithin a biosensor matrix, for example using the BIAcore® system(BIAcore International AB, a GE Healthcare company, Uppsala, Sweden andPiscataway, N.J., US). For further descriptions, see Jonsson et al.,Ann. Biol. Clin., 51: 19-26 (1993); Jonsson et al. BioTechniques, 11:620-627 (1991); Johnsson et al., J. Mol. Recognit., 8: 125-131 (1995);and Johnsson et al., Anal. Biochem., 198: 268-277 (1991).

The term “K_(on)”, as used herein, is intended to refer to the on rateconstant for association of a binding protein (e.g., an antibody) to acognate partner (e.g., an antigen) to form a binding partner/cognatepartner (e.g., antibody/antigen) complex as is known in the art. The“K_(on)” also is known by the terms “association rate constant,” or“k_(a),” as used interchangeably herein. This value indicating thebinding rate of an antibody to its target antigen or the rate of complexformation between an antibody and antigen also is shown by the equation:

Antibody (“Ab”)+Antigen (“Ag”)→Ab-Ag.

The term “K_(off),” as used herein, is intended to refer to the off rateconstant for dissociation of a binding protein (e.g., an antibody) fromthe, e.g., antibody/antigen complex as is known in the art. The“K_(off)” also is known by the terms “dissociation rate constant” or“k_(d)” as used interchangeably herein. This value indicates thedissociation rate of an antibody from its target antigen or separationof Ab-Ag complex over time intofree antibody and antigen as shown by theequation below:

Ab+Ag←Ab-Ag.

The terms “equilibrium dissociation constant” or “K_(D)”, as usedinterchangeably herein, refer to the value obtained in a titrationmeasurement at equilibrium, or by dividing the dissociation rateconstant (K_(off)) by the association rate constant (K_(on)). Theassociation rate constant, the dissociation rate constant, and theequilibrium dissociation constant are used to represent the bindingaffinity of an antibody to an antigen. Methods for determiningassociation and dissociation rate constants are well known in the art.Using fluorescence-based techniques offers high sensitivity and theability to examine samples in physiological buffers at equilibrium.Other experimental approaches and instruments such as a BIAcore® surfaceplasmon resonance (biomolecular interaction analysis) assay can be used(e.g., instrument available from BIAcore International AB, a GEHealthcare company, Uppsala, Sweden). Additionally, a KinExA® (KineticExclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.)can also be used.

“Label” and “detectable label” mean a moiety attached to a specificbinding partner, such as an antibody or an analyte bound by theantibody, e.g., to render the reaction between members of a specificbinding pair, such as an antibody and an analyte, detectable. Thespecific binding partner, e.g., antibody or analyte, so labeled isreferred to as “detectably labeled”. Thus, the term “labeled bindingprotein” as used herein, refers to a protein with a label incorporatedthat provides for the identification of the binding protein. In anembodiment, the label is a detectable marker that can produce a signalthat is detectable by visual or instrumental means, e.g., incorporationof a radiolabeled amino acid or attachment to a polypeptide of biotinylmoieties that can be detected by a marked avidin (e.g., an avidin or astreptavidin containing a fluorescent marker or enzymatic activity thatcan be detected by optical or colorimetric methods). Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, an ¹⁵³Sm); chromogens, fluorescent labels(e.g., FITC, rhodamine, and lanthanide phosphors), enzymatic labels(e.g., horseradish peroxidase, luciferase, alkaline phosphatase);chemiluminescent markers; biotinyl groups; predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, and epitope tags); and magnetic agents, such as gadoliniumchelates. Representative examples of labels commonly employed forimmunoassays include moieties that produce light, e.g., acridiniumcompounds, and moieties that produce fluorescence, e.g., fluorescein.Other labels are known in the art or described herein. In this regard,the moiety itself may not be detectably labeled but may becomedetectable upon reaction with yet another moiety. Use of “detectablylabeled” is intended to encompass the latter type of detectablelabeling.

The term “antibody conjugate” refers to a binding protein, such as anantibody, chemically linked to a second chemical moiety, such as atherapeutic or cytotoxic agent. The term “agent” is used herein todenote a chemical compound, a mixture of chemical compounds, abiological macromolecule, or an extract made from biological materials.Preferably, the therapeutic or cytotoxic agents include, but are notlimited to, pertussis toxin, taxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicine, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof.

The terms “crystal” and “crystallized” as used herein, refer to abinding protein (e.g., an antibody), or antigen binding portion thereof,that exists in the form of a crystal. Crystals are one form of the solidstate of matter, which is distinct from other forms such as theamorphous solid state or the liquid crystalline state. Crystals arecomposed of regular, repeating, three-dimensional arrays of atoms, ions,molecules (e.g., proteins such as antibodies), or molecular assemblies(e.g., antigen/antibody complexes, including Fab/antigen complexes).These three-dimensional arrays are arranged according to specificmathematical relationships that are well-understood in the field. Thefundamental unit, or building block, that is repeated in a crystal iscalled the asymmetric unit. Repetition of the asymmetric unit in anarrangement that conforms to a given, well-defined crystallographicsymmetry provides the “unit cell” of the crystal. Repetition of the unitcell by regular translations in all three dimensions provides thecrystal. See, Giege et al., In Crystallization of Nucleic Acids andProteins, a Practical Approach, 2nd ed., (Ducruix and Giege, eds.)(Oxford University Press, New York, 1999), chapter 1, pages 1-16.

The term “polynucleotide” means a polymeric form of two or morenucleotides, either ribonucleotides or deoxyribonucleotides or amodified form of either type of nucleotide. The term includes single anddouble stranded forms of DNA.

The term “isolated polynucleotide” shall mean a polynucleotide (e.g., ofgenomic, cDNA, or synthetic origin, or some combination thereof) that,by virtue of its origin, the “isolated polynucleotide” is not associatedwith all or a portion of a polynucleotide with which the “isolatedpolynucleotide” is found in nature; is operably linked to apolynucleotide that it is not linked to in nature; or does not occur innature as part of a larger sequence.

The term “vector,” is intended to refer to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions. RNA versions of vectors(including RNA viral vectors) may alsofind use in the invention.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them tofunction intheir intended manner. A control sequence “operably linked” to a codingsequence is ligated in such a way that expression of the coding sequenceis achieved under conditions compatible with the control sequences.“Operably linked” sequences include expression control sequences thatare contiguous with a gene of interest, expression control sequencesthat act in trans, i.e., located on a different nucleic acid moleculethan a gene of interest, as well as expression control sequences thatare located on the same nucleic acid molecule as, but at a distancefrom, a gene of interest. The term “expression control sequence” as usedherein refers to polynucleotide sequences which are necessary to effectthe expression and processing of coding sequences to which they areligated. Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include a promoter, a ribosomal binding site, and atranscription termination sequence; in eukaryotes, generally, suchcontrol sequences include a promoter and a transcription terminationsequence. The term “control sequences” is intended to include componentswhose presence is essential for expression and processing, and can alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences.

“Transformation,” refers to any process by which exogenous nucleic acid(e.g., a DNA molecule) enters a host cell. Transformation may occurunder natural or artificial conditions using various methods well knownin the art. Transformation may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method is selected based on the host cellbeing transformed and may include, but is not limited to, plasmid uptakeacross a cellular membrane, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells which transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), is intended torefer to a cell into which exogenous DNA has been introduced. In anembodiment, the host cell comprises two or more (e.g., multiple) nucleicacids encoding antibodies, such as, by way of non-limiting example, thehost cells described in U.S. Pat. No. 7,262,028. Such terms are intendedto refer not only to the particular subject cell, but, also to theprogeny of such a cell. Because certain modifications may occur insucceeding generations due to either mutation or environmentalinfluences, such progeny may not, in fact, be identical to the parentcell, but are still included within the scope of the term “host cell” asused herein. In an embodiment, host cells include prokaryotic andeukaryotic cells selected from any of the Kingdoms of life. In anotherembodiment, eukaryotic cells include protist, fungal, plant and animalcells. In another embodiment, host cells include but are not limited toprokaryotic species, such Escherichia coli; mammalian cell lines, suchas CHO, HEK 293, COS, NS0, SP2, and PER.C6; the insect cell line Sf9;and fungal cell species, such as Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, second ed. (Cold Spring Harbor Laboratory Press, ColdSpring Harbor, 1989).

“Transgenic organism,” as known in the art, refers to an organism havingcells that contain a transgene, wherein the transgene introduced intothe organism (or an ancestor of the organism) expresses a polypeptidenot naturally expressed in the organism. A “transgene” is a DNAconstruct, which is stably and operably integrated into the genome of acell from which a transgenic organism develops, directing the expressionof an encoded gene product in one or more cell types or tissues of thetransgenic organism.

The term “regulate” and “modulate” are used interchangeably, and, asused herein, refers to a change or an alteration in the activity of amolecule of interest (e.g., the biological activity of hDLL4).Modulation may be an increase or a decrease in the magnitude of acertain activity or function of the molecule of interest. Exemplaryactivities and functions of a molecule include, but are not limited to,binding characteristics, enzymatic activity, cell receptor activation,and signal transduction.

Correspondingly, the term “modulator,” as used herein, is a compoundcapable of changing or altering an activity or function of a molecule ofinterest (e.g., the biological activity of hDLL4). For example, amodulator may cause an increase or decrease in the magnitude of acertain activity or function of a molecule compared to the magnitude ofthe activity or function observed in the absence of the modulator. Incertain embodiments, a modulator is an inhibitor, which decreases themagnitude of at least one activity or function of a molecule. Exemplaryinhibitors include, but are not limited to, proteins, peptides,antibodies, peptibodies, carbohydrates or small organic molecules.Peptibodies have been described. See, e.g., PCT Publication No.WO01/83525.

The term “agonist”, as used herein, refers to a modulator that, whencontacted with a molecule of interest, causes an increase in themagnitude of a certain activity or function of the molecule compared tothe magnitude of the activity or function observed in the absence of theagonist. Particular agonists of interest may include, but are notlimited to, members of the Notch-signaling pathway, DLL4 polypeptidesand nucleic acids, carbohydrates, or any other molecules that bind toDLL4.

The term “antagonist” or “inhibitor”, as used herein, refers to amodulator that, when contacted with a molecule of interest causes adecrease in the magnitude of a certain activity or function of themolecule compared to the magnitude of the activity or function observedin the absence of the antagonist. Particular antagonists of interestinclude those that block or modulate the biological or immunologicalactivity of DLL4, especially human DLL4 (hDLL4). Antagonists andinhibitors of hDLL4 may include, but are not limited to, proteins,nucleic acids, carbohydrates, or any other molecule, which binds tohDLL4 and/or rodent DLL4.

As used herein, the term “effective amount” refers to the amount of atherapy that is sufficient to reduce or ameliorate the severity and/orduration of a disorder or one or more symptoms thereof; inhibit orprevent the advancement of a disorder; cause regression of a disorder;inhibit or prevent the recurrence, development, onset, or progression ofone or more symptoms associated with a disorder; detect a disorder; orenhance or improve the prophylactic or therapeutic effect(s) of anothertherapy (e.g., prophylactic or therapeutic agent).

“Patient” and “subject” may be used interchangeably herein to refer toan animal, such as a mammal, including a primate (for example, a human,a monkey, and a chimpanzee), a non-primate (for example, a cow, a pig, acamel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guineapig, a cat, a dog, a rat, a mouse, and a whale), a bird (e.g., a duck ora goose), and a shark. Preferably, a patient or subject is a human, suchas a human being treated or assessed for a disease, disorder, orcondition; a human at risk for a disease, disorder, or condition; ahuman having a disease, disorder, or condition; and/or human beingtreated for a disease, disorder, or condition. More preferably, apatient or subject is being treated or assessed for cancer or otherdisease in which the existing aberrant DLL4 expression supports thecancer or other disease and inhibition or disruption of DLL4 activity isdesirable to treat the cancer or other disease.

The term “sample,” as used herein, is used in its broadest sense. A“biological sample,” as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans, mice,rats, monkeys, dogs, rabbits and other animals. Such substances include,but are not limited to, blood, (e.g., whole blood), plasma, serum,urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes,monocytes, other cells, organs, tissues, bone marrow, lymph nodes andspleen.

“Component,” “components,” and “at least one component,” refer generallyto a capture antibody, a detection or conjugate antibody, a control, acalibrator, a series of calibrators, a sensitivity panel, a container, abuffer, a diluent, a salt, an enzyme, a co-factor for an enzyme, adetection reagent, a pretreatment reagent/solution, a substrate (e.g.,as a solution), a stop solution, and the like that can be included in akit for assay of a test sample, such as a patient urine, serum or plasmasample, in accordance with the methods described herein and othermethods known in the art. Thus, in the context of the presentdisclosure, “at least one component,” “component,” and “components” caninclude a polypeptide or other analyte as above, such as a compositioncomprising an analyte such as a polypeptide, which is optionallyimmobilized on a solid support, such as by binding to an anti-analyte(e.g., anti-polypeptide) antibody. Some components can be in solution orlyophilized for reconstitution for use in an assay.

“Risk” refers to the possibility or probability of a particular eventoccurring either presently or at some point in the future. “Riskstratification” refers to an array of known clinical risk factors thatallows physicians to classify patients into a low, moderate, high orhighest risk of developing a particular disease, disorder or condition.

“Specific” and “specificity” in the context of an interaction betweenmembers of a specific binding pair (e.g., an antigen or fragment thereofand an antibody or antigen binding fragment thereof) refer to theselective reactivity of the interaction. The phrase “specifically bindsto” and analogous phrases refer to the ability of binding proteins, suchas antibodies (or antigen binding fragments thereof), to bindspecifically to a molecule of interest (or a fragment thereof) and notbind specifically to other entities.

“Specific binding partner” is a member of a specific binding pair. Aspecific binding pair comprises two different molecules, whichspecifically bind to each other through chemical or physical means.Therefore, in addition to antigen and antibody specific binding pairs,other specific binding pairs can include biotin and avidin (orstreptavidin), carbohydrates and lectins, complementary nucleotidesequences, effector and receptor molecules, cofactors and enzymes,enzyme inhibitors and enzymes, and the like. Furthermore, specificbinding pairs can include members that are analogs of the originalspecific binding members, for example, an analyte-analog. Immunoreactivespecific binding members include antigens, antigen fragments, andantibodies, including monoclonal and polyclonal antibodies as well ascomplexes, fragments, and variants (including fragments of variants)thereof, whether isolated or recombinantly produced.

“Variant” as used herein means a polypeptide that differs from a givenpolypeptide (e.g., DLL4 polypeptide or anti-DLL4 antibody) in amino acidsequence by the addition (e.g., insertion), deletion, or conservativesubstitution of amino acids, but that retains the biological activity ofthe given polypeptide (e.g., a variant DLL4 may compete with a wildtypeDLL4 for binding with an anti-DLL4 antibody if the variant DLL4 retainsthe original antibody binding site (epitope) of the wildtype DLL4). Aconservative substitution of an amino acid, i.e., replacing an aminoacid with a different amino acid of similar properties (e.g.,hydrophilicity and degree and distribution of charged regions) isrecognized in the art as typically involving a minor change. These minorchanges can be identified, in part, by considering the hydropathic indexof amino acids, as understood in the art (see, e.g., Kyte et al., J.Mol. Biol., 157: 105-132 (1982)). The hydropathic index of an amino acidis based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids also can be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity (see, e.g., U.S. Pat. No.4,554,101). Substitution of amino acids having similar hydrophilicityvalues can result in peptides retaining biological activity, for exampleimmunogenicity, as is understood in the art. In one aspect,substitutions are performed with amino acids having hydrophilicityvalues within ±2 of each other. Both the hydrophobicity index and thehydrophilicity value of amino acids are influenced by the particularside chain of that amino acid. Consistent with that observation, aminoacid substitutions that are compatible with biological function areunderstood to depend on the relative similarity of the amino acids, andparticularly the side chains of those amino acids, as revealed by thehydrophobicity, hydrophilicity, charge, size, and other properties.“Variant” also can be used to describe a polypeptide or fragment thereofthat has been differentially processed, such as by proteolysis,phosphorylation, or other post-translational modification, yet retainsits biological activity or antigen reactivity, e.g., the ability to bindto DLL4. Use of “variant” herein is intended to encompass fragments of avariant unless otherwise contradicted by context.

The term “sample”, as used herein, is used in its broadest sense. A“biological sample”, as used herein, includes, but is not limited to,any quantity of a substance from a living thing or formerly livingthing. Such living things include, but are not limited to, humans, mice,rats, monkeys, dogs, rabbits and other animals. Such substances include,but are not limited to, blood, serum, urine, synovial fluid, cells,organs, tissues, bone marrow, lymph nodes, and spleen.

I. Antibodies that Bind Human DLL4.

One aspect of the present invention provides isolated rat monoclonalantibodies, or antigen-binding portions thereof, that bind to DLL4 withhigh affinity, a slow off rate, and/or high neutralizing capacity.Another aspect of the invention provides chimeric antibodies that bindDLL4. In another aspect, the invention provides CDR grafted antibodies,or antigen-binding portions thereof, that DLL4. Another aspect of theinvention provides humanized antibodies, or antigen-binding portionsthereof, that bind DLL4. In an embodiment, the antibodies, or portionsthereof, are isolated antibodies or isolated portions thereof. Inanother embodiment, the antibodies, or antigen-binding portions thereof,of the invention are neutralizing anti-DLL antibodies. Advantageously,such antibodies or antigen-binding portions thereof that bind DLL4 finduse as therapeutic agents that can be administered to an individual(human or other mammal). Preferably, the antibodies or antigen-bindingportions thereof of the invention are neutralizing anti-DLL4 and/oranti-VEGFR2 antibodies.

A. Method of Making Anti-DLL4 Antibodies.

Antibodies of the present invention may be made by any of a number oftechniques known in the art. Aspects of various techniques that may beemployed to obtain DLL4 monoclonal antibodies according to the inventionare described below.

1. Anti-DLL4 Monoclonal Antibodies Using Hybridoma Technology.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, second edition, (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, 1988); Hammerling, et al., In MonoclonalAntibodies and T-Cell Hybridomas, (Elsevier, New York, 1981). It is alsonoted that the term “monoclonal antibody” as used herein is not limitedto antibodies produced through hybridoma technology. The term“monoclonal antibody” refers to an antibody that is derived from asingle clone, including any eukaryotic, prokaryotic, or phage clone, andnot the method by which it is produced.

In an embodiment, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from an animal, e.g., a rat or a mouse, immunizedwith DLL4 with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention. Briefly, rats can be immunized witha DLL4 antigen (see, Examples, below). In a preferred embodiment, theDLL4 antigen is administered with an adjuvant to stimulate the immuneresponse. Such adjuvants include complete or incomplete Freund'sadjuvant, RIBI (muramyl dipeptides) or ISCOM (immunostimulatingcomplexes). Such adjuvants may protect the polypeptide from rapiddispersal by sequestering it in a local deposit, or they may containsubstances that stimulate the host to secrete factors that arechemotactic for macrophages and other components of the immune system.Preferably, if a polypeptide is being administered, the immunizationschedule will involve two or more administrations of the polypeptide,spread out over several weeks; however, a single administration of thepolypeptide may also be used.

After immunization of an animal with a DLL4 antigen, antibodies and/orantibody-producing cells may be obtained from the animal. An anti-DLL4antibody-containing serum is obtained from the animal by bleeding orsacrificing the animal. The serum may be used as it is obtained from theanimal, an immunoglobulin fraction may be obtained from the serum, orthe anti-DLL4 antibodies may be purified from the serum. Serum orimmunoglobulins obtained in this manner are polyclonal, thus having aheterogeneous array of properties.

Once an immune response is detected, e.g., antibodies specific for theantigen DLL4 are detected in the rat serum, the rat spleen is harvestedand splenocytes isolated. The splenocytes are then fused by well-knowntechniques to any suitable myeloma cells, for example cells from cellline SP20 available from the American Type Culture Collection (ATCC,Manassas, Va., US). Hybridomas are selected and cloned by limiteddilution. The hybridoma clones are then assayed by methods known in theart for cells that secrete antibodies capable of binding DLL4. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by immunizing rats with positive hybridoma clones.

In another embodiment, antibody-producing immortalized hybridomas may beprepared from the immunized animal. After immunization, the animal issacrificed and the splenic B cells are fused to immortalized myelomacells as is well known in the art. See, e.g., Harlow and Lane, supra. Ina preferred embodiment, the myeloma cells do not secrete immunoglobulinpolypeptides (a non-secretory cell line). After fusion and antibioticselection, the hybridomas are screened using DLL4, or a portion thereof,or a cell expressing DLL4. In a preferred embodiment, the initialscreening is performed using an enzyme-linked immunosorbent assay(ELISA) or a radioimmunoassay (RIA), preferably an ELISA. An example ofELISA screening is provided in PCT Publication No. WO 00/37504.

Anti-DLL4 antibody-producing hybridomas are selected, cloned, andfurther screened for desirable characteristics, including robusthybridoma growth, high antibody production, and desirable antibodycharacteristics, as discussed further below. Hybridomas may be culturedand expanded in vivo in syngeneic animals, in animals that lack animmune system, e.g., nude mice, or in cell culture in vitro. Methods ofselecting, cloning and expanding hybridomas are well known to those ofordinary skill in the art.

In a preferred embodiment, hybridomas are rat hybridomas, as describedherein. In another embodiment, hybridomas are produced in a non-human,non-rat species such as mice, sheep, pigs, goats, cattle, or horses. Inyet another preferred embodiment, the hybridomas are human hybridomas,in which a human non-secretory myeloma is fused with a human cellexpressing an anti-DLL4 antibody.

Antibody fragments that recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)2 fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce two identical Fabfragments) or pepsin (to produce a F(ab′)₂ fragment). A F(ab′)₂ fragmentof an IgG molecule retains the two antigen-binding sites of the larger(“parent”) IgG molecule, including both light chains (containing thevariable light chain and constant light chain regions), the CH1 domainsof the heavy chains, and a disulfide-forming hinge region of the parentIgG molecule. Accordingly, a F(ab′)2 fragment is still capable ofcrosslinking antigen molecules like the parent IgG molecule.

2. Anti-DLL4 Monoclonal Antibodies Using SLAM.

In another aspect of the invention, recombinant antibodies are generatedfrom single, isolated lymphocytes using a procedure referred to in theart as the selected lymphocyte antibody method (SLAM), as described inU.S. Pat. No. 5,627,052; PCT Publication No. WO 92/02551; and Babcook etal., Proc. Natl. Acad. Sci. USA, 93: 7843-7848 (1996). In this method,single cells secreting antibodies of interest, e.g., lymphocytes derivedfrom any one of the immunized animals described in Section I.A.1(above), are screened using an antigen-specific hemolytic plaque assay,wherein the antigen DLL4, a subunit of DLL4, or a fragment thereof, iscoupled to sheep red blood cells using a linker, such as biotin, andused to identify single cells that secrete antibodies with specificityfor DLL4. Following identification of antibody-secreting cells ofinterest, heavy- and light-chain variable region cDNAs are rescued fromthe cells by reverse transcriptase-PCR (RT-PCR) and these variableregions can then be expressed, in the context of appropriateimmunoglobulin constant regions (e.g., human constant regions), inmammalian host cells, such as COS or CHO cells. The host cellstransfected with the amplified immunoglobulin sequences, derived from invivo selected lymphocytes, can then undergofurther analysis andselection in vitro, for example, by panning the transfected cells toisolate cells expressing antibodies to DLL4. The amplifiedimmunoglobulin sequences further can be manipulated in vitro, such as byin vitro affinity maturation method. See, for example, PCT PublicationNo. WO 97/29131 and PCT Publication No. WO 00/56772.

3. Anti-DLL4 Monoclonal Antibodies Using Transgenic Animals.

In another embodiment of the instant invention, antibodies are producedby immunizing a non-human animal comprising some, or all, of the humanimmunoglobulin locus with a DLL4 antigen. In an embodiment, thenon-human animal is a XENOMOUSE® transgenic mouse, an engineered mousestrain that comprises large fragments of the human immunoglobulin lociand is deficient in mouse antibody production. See, e.g., Green et al.,Nature Genetics, 7: 13-21 (1994) and U.S. Pat. Nos. 5,916,771;5,939,598; 5,985,615; 5,998,209; 6,075,181; 6,091,001; 6,114,598; and6,130,364. See also PCT Publication Nos. WO 91/10741; WO 94/02602; WO96/34096; WO 96/33735; WO 98/16654; WO 98/24893; WO 98/50433; WO99/45031; WO 99/53049; WO 00/09560; and WO 00/37504. The XENOMOUSE®transgenic mouse produces an adult-like human repertoire of fully humanantibodies, and generates antigen-specific human monoclonal antibodies.The XENOMOUSE® transgenic mouse contains approximately 80% of the humanantibody repertoire through introduction of megabase sized, germlineconfiguration YAC fragments of the human heavy chain loci and x lightchain loci. See Mendez et al., Nature Genetics, 15: 146-156 (1997),Green and Jakobovits, J. Exp. Med., 188: 483-495 (1998), the disclosuresof which are hereby incorporated by reference.

4. Anti-DLL4 Monoclonal Antibodies Using Recombinant Antibody Libraries.

In vitro methods also can be used to make the antibodies of theinvention, wherein an antibody library is screened to identify anantibody having the desired DLL4-binding specificity. Methods for suchscreening of recombinant antibody libraries are well known in the artand include methods described in, for example, U.S. Pat. No. 5,223,409(Ladner et al.); PCT Publication No. WO 92/18619 (Kang et al.); PCTPublication No. WO 91/17271 (Dower et al.); PCT Publication No. WO92/20791 (Winter et al.); PCT Publication No. WO 92/15679 (Markland etal.); PCT Publication No. WO 93/01288 (Breitling et al.); PCTPublication No. WO 92/01047 (McCafferty et al.); PCT Publication No. WO92/09690 (Garrard et al.); Fuchs et al., Bio/Technology, 9: 1369-1372(1991); Hay et al., Hum. Antibod. Hybridomas, 3: 81-85 (1992); Huse etal., Science, 246: 1275-1281 (1989); McCafferty et al., Nature, 348:552-554 (1990); Griffiths et al., EMBO J., 12: 725-734 (1993); Hawkinset al., J. Mol. Biol., 226: 889-896 (1992); Clackson et al., Nature,352: 624-628 (1991); Gram et al., Proc. Natl. Acad. Sci. USA, 89:3576-3580 (1992); Garrard et al., Bio/Technology, 9: 1373-1377 (1991);Hoogenboom et al., Nucl. Acids Res., 19: 4133-4137 (1991); Barbas etal., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991); US PatentApplication Publication No. 2003/0186374; and PCT Publication No. WO97/29131, the contents of each of which are incorporated herein byreference.

The recombinant antibody library may be from a subject immunized withDLL4, or a portion of DLL4. Alternatively, the recombinant antibodylibrary may be from a naïve subject, i.e., one who has not beenimmunized with DLL4, such as a human antibody library from a humansubject who has not been immunized with human DLL4. Antibodies of theinvention are selected by screening the recombinant antibody librarywith the peptide comprising human DLL4 to thereby select thoseantibodies that recognize DLL4. Methods for conducting such screeningand selection are well known in the art, such as described in thereferences in the preceding paragraph. To select antibodies of theinvention having particular binding affinities for DLL4, such as thosethat dissociate from human DLL4 with a particular K_(off) rate constant,the art-known method of surface plasmon resonance can be used to selectantibodies having the desired K_(off) rate constant. To selectantibodies of the invention having a particular neutralizing activityfor hDLL4, such as those with a particular IC₅₀, standard methods knownin the art for assessing the inhibition of DLL4 activity may be used.

In one aspect, the invention pertains to an isolated antibody, or anantigen-binding portion thereof, that binds human DLL4. Preferably, theantibody is a neutralizing antibody. In various embodiments, theantibody is a recombinant antibody or a monoclonal antibody.

For example, antibodies of the present invention can also be generatedusing various phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles which carry the polynucleotide sequences encoding them.Such phage can be utilized to display antigen-binding domains expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Phage expressing an antigen binding domain that binds theantigen of interest can be selected or identified with antigen, e.g.,using labeled antigen or antigen bound or captured to a solid surface orbead. Phage used in these methods are typically filamentous phageincluding fd and M13 binding domains expressed from phage with Fab, Fv,or disulfide stabilized Fv antibody domains recombinantly fused toeither the phage gene III or gene VIII protein. Examples of phagedisplay methods that can be used to make the antibodies of the presentinvention include those disclosed in Brinkmann et al., J. Immunol.Methods, 182: 41-50 (1995); Ames et al., J. Immunol. Methods,184:177-186 (1995); Kettleborough et al., Eur. J. Immunol., 24: 952-958(1994); Persic et al., Gene, 187: 9-18 (1997); Burton et al., Advancesin Immunology, 57: 191-280 (1994); PCT Publication No. WO 92/01047; PCTPublication Nos. WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426;5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743; and5,969,108.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies including human antibodies or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′, and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax et al., BioTechniques, 12(6):864-869 (1992); Sawai et al., Am. J. Reprod. Immunol., 34: 26-34 (1995);and Better et al., Science, 240: 1041-1043 (1988). Examples oftechniques which can be used to produce single-chain Fvs and antibodiesinclude those described in U.S. Pat. Nos. 4,946,778 and 5,258,498;Huston et al., Methods in Enzymology, 203: 46-88 (1991); Shu et al.,Proc. Natl. Acad. Sci. USA, 90: 7995-7999 (1993); and Skerra et al.,Science, 240: 1038-1041 (1988).

Alternative to screening of recombinant antibody libraries by phagedisplay, other methodologies known in the art for screening largecombinatorial libraries can be applied to the identification ofantibodies of the invention. One type of alternative expression systemis one in which the recombinant antibody library is expressed asRNA-protein fusions, as described in PCT Publication No. WO 98/31700(Szostak and Roberts), and in Roberts and Szostak, Proc. Natl. Acad.Sci. USA, 94: 12297-12302 (1997). In this system, a covalent fusion iscreated between an mRNA and the peptide or protein that it encodes by invitro translation of synthetic mRNAs that carry puromycin, a peptidylacceptor antibiotic, at their 3′ end. Thus, a specific mRNA can beenriched from a complex mixture of mRNAs (e.g., a combinatorial library)based on the properties of the encoded peptide or protein, e.g.,antibody, or portion thereof, such as binding of the antibody, orportion thereof, to the dual specificity antigen. Nucleic acid sequencesencoding antibodies, or portions thereof, recovered from screening ofsuch libraries can be expressed by recombinant means as described above(e.g., in mammalian host cells) and, moreover, can be subjectedtofurther affinity maturation by either additional rounds of screeningof mRNA-peptide fusions in which mutations have been introduced into theoriginally selected sequence(s), or by other methods for affinitymaturation in vitro of recombinant antibodies, as described above. Apreferred example of this methodology, is the PROfusion displaytechnology employed in the Examples (infra).

In another approach the antibodies of the present invention can also begenerated using yeast display methods known in the art. In yeast displaymethods, genetic methods are used to tether antibody domains to theyeast cell wall and display them on the surface of yeast. In particular,such yeast can be utilized to display antigen-binding domains expressedfrom a repertoire or combinatorial antibody library (e.g., human ormurine). Examples of yeast display methods that can be used to make theantibodies of the present invention include those disclosed in U.S. Pat.No. 6,699,658 (Wittrup et al.) incorporated herein by reference.

B. Production of Recombinant DLL4 Antibodies

Antibodies of the present invention may be produced by any of a numberof techniques known in the art. For example, expression from host cells,wherein expression vector(s) encoding the heavy and light chains is(are) transfected into a host cell by standard techniques. The variousforms of the term “transfection” are intended to encompass a widevariety of techniques commonly used for the introduction of exogenousDNA into a prokaryotic or eukaryotic host cell, e.g., electroporation,calcium-phosphate precipitation, DEAE-dextran transfection and the like.Although it is possible to express the antibodies of the invention ineither prokaryotic or eukaryotic host cells, expression of antibodies ineukaryotic cells is preferable, and most preferable in mammalian hostcells, because such eukaryotic cells (and in particular mammalian cells)are more likely than prokaryotic cells to assemble and secrete aproperly folded and immunologically active antibody.

Exemplary mammalian host cells for expressing the recombinant antibodiesof the invention include Chinese Hamster Ovary (CHO cells) (includingdhfr− CHO cells, described in Urlaub and Chasin, Proc. Natl. Acad. Sci.USA, 77: 4216-4220 (1980), used with a DHFR selectable marker, e.g., asdescribed in Kaufman and Sharp, J. Mol. Biol., 159: 601-621 (1982), NS0myeloma cells, COS cells, and SP2 cells. When recombinant expressionvectors encoding antibody genes are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown. Antibodies can berecovered from the culture medium using standard protein purificationmethods.

Host cells can also be used to produce functional antibody fragments,such as Fab fragments or scFv molecules. It will be understood thatvariations on the above procedure are within the scope of the presentinvention. For example, it may be desirable to transfect a host cellwith DNA encoding functional fragments of either the light chain and/orthe heavy chain of an antibody of this invention. Recombinant DNAtechnology may also be used to remove some, or all, of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to the antigens of interest. The molecules expressed from suchtruncated DNA molecules are also encompassed by the antibodies of theinvention. In addition, bifunctional antibodies may be produced in whichone heavy and one light chain are an antibody of the invention (i.e.,binds human DLL4) and the other heavy and light chain are specific foran antigen other than human DLL4 by crosslinking an antibody of theinvention to a second antibody by standard chemical crosslinkingmethods.

In a preferred system for recombinant expression of an antibody, orantigen-binding portion thereof, of the invention, a recombinantexpression vector encoding both the antibody heavy chain and theantibody light chain is introduced into dhfr− CHO cells by calciumphosphate-mediated transfection. Within the recombinant expressionvector, the antibody heavy and light chain genes are each operativelylinked to CMV enhancer/AdMLP promoter regulatory elements to drive highlevels of transcription of the genes. The recombinant expression vectoralso carries a DHFR gene, which allows for selection of CHO cells thathave been transfected with the vector using methotrexateselection/amplification. The selected transformant host cells arecultured to allow for expression of the antibody heavy and light chainsand intact antibody is recovered from the culture medium. Standardmolecular biology techniques are used to prepare the recombinantexpression vector, transfect the host cells, select for transformants,culture the host cells and recover the antibody from the culture medium.Still further the invention provides a method of synthesizing arecombinant antibody of the invention by culturing a host cell of theinvention in a suitable culture medium until a recombinant antibody ofthe invention is synthesized. The method can further comprise isolatingthe recombinant antibody from the culture medium.

1. Anti-DLL4 Antibodies.

Amino acid sequences of VH and VL regions of isolated rat monoclonalantibodies that bind human DLL4 are shown for clones 38H12, 1A11, 37D10,32C7, 14G1, 14A11, and 15D6 in Table 9 (See, Example 4, below). Theisolated anti-DLL4 antibody CDR sequences described herein establish afamily of DLL4 binding proteins, isolated in accordance with thisinvention, and comprising polypeptides that include the CDR sequencesderived therefrom and affinity matured clones thereof. Sequences ofvariable regions and CDRs of the monoclonal antibodies and affinitymatured derivatives thereof are listed in Tables 9, 11, 16, 20, and 21.To generate and to select CDRs for binding proteins according to theinvention having preferred DLL4 binding and/or neutralizing activitywith respect to human DLL4, standard methods known in the art forgenerating binding proteins of the present invention and assessing theDLL4 binding and/or neutralizing characteristics of those bindingprotein may be used, including but not limited to those specificallydescribed herein.

Based on an alignment of the amino acid sequences of the CDRs of theheavy chain variable regions (VH) and the light chain variable regions(VL) of the anti-DLL4 antibody clones described herein, the inventionprovides a DLL4 binding protein comprising an antigen binding domaincapable of binding human DLL4, said antigen binding domain comprising atleast one or more of the six CDRs, i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1,CDR-L2, and CDRL-3, defined below:

CDR-H1 is selected from the group consisting of:

-   -   X₁—X₂—X₃—X₄—X₅ (SEQ ID NO:151), wherein;        -   X₁ is N, H, or Y;        -   X₂ is F;        -   X₃ is P;        -   X₄ is M; and        -   X₅ is A or S;    -   residues 31-35 of SEQ ID NO:157 (CDR-H1 38H12);    -   residues 31-35 of SEQ ID NO:161 (CDR-H1 37D10);    -   residues 31-35 of SEQ ID NO:163 (CDR-H1 32C7);    -   residues 31-35 of SEQ ID NO:165 (CDR-H1 14G1);    -   residues 31-35 of SEQ ID NO:167 (CDR-H1 14A11);    -   residues 31-35 of SEQ ID NO:169 (CDR-H1 15D6);    -   residues 31-35 of SEQ ID NO:171 (CDR-H1 VH.1 1A11);    -   residues 31-35 of SEQ ID NO:172 (CDR-H1 VH.1a 1A11);    -   residues 31-35 of SEQ ID NO:173 (CDR-H1 VH.1b 1A11);    -   residues 31-35 of SEQ ID NO:174 (CDR-H1 VH.2a 1A11);    -   residues 31-35 of SEQ ID NO:179 (CDR-H1 VH.1 38H12);    -   residues 31-35 of SEQ ID NO:180 (CDR-H1 VH.1A 38H12);    -   residues 31-35 of SEQ ID NO:181 (CDR-H1 VH.1b 38H12);    -   residues 31-35 of SEQ ID NO:182 (CDR-H1 VH.2a 38H12);    -   residues 31-35 of SEQ ID NO:187 (CDR-H1 h1A11VH.1);    -   residues 31-35 of SEQ ID NO:188 (CDR-H1 h1A11.A6);    -   residues 31-35 of SEQ ID NO:189 (CDR-H1 h1A11.A8);    -   residues 31-35 of SEQ ID NO:190 (CDR-H1 h1A11.C6);    -   residues 31-35 of SEQ ID NO:191 (CDR-H1 h1A11.A11);    -   residues 31-35 of SEQ ID NO:192 (CDR-H1 h1A11.B5);    -   residues 31-35 of SEQ ID NO:193 (CDR-H1 h1A11.E12);    -   residues 31-35 of SEQ ID NO:194 (CDR-H1 h1A11.G3);    -   residues 31-35 of SEQ ID NO:195 (CDR-H1 h1A11.F5); and    -   residues 31-35 of SEQ ID NO:196 (CDR-H1 h1A11.H2);

CDR-H2 is selected from the group consisting of:

-   -   X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—X₉—X₁₀—X₁₁—X₁₂—X₁₃—X₁₄—X₁₅—X₁₆—X₁₇ (SEQ        ID NO:152), wherein;        -   X₁ is T or S;        -   X₂ is I;        -   X₃ is S;        -   X₄ is S or G;        -   X₅ is S;        -   X₆ is D;        -   X₇ is G, A, D, S, or E;        -   X₈ is T or W;        -   X₉ is T, P, or A;        -   X₁₀ is Y, S, T, or N;        -   X₁₁ is Y or I;        -   X₁₂ is R or G;        -   X₁₃ is D;        -   X₁₄ is 5;        -   X₁₅ is V;        -   X₁₆ is K; and        -   X₁₇ is G;    -   residues 50-66 of SEQ ID NO:157 (CDR-H2 38H12);    -   residues 50-68 of SEQ ID NO:161 (CDR-H2 37D10);    -   residues 50-66 of SEQ ID NO:163 (CDR-H2 32C7);    -   residues 50-66 of SEQ ID NO:165 (CDR-H2 14G1);    -   residues 50-66 of SEQ ID NO:167 (CDR-H2 14A11);    -   residues 50-66 of SEQ ID NO:169 (CDR-H2 15D6);    -   residues 50-66 of SEQ ID NO:171 (CDR-H2 VH.1 1A11);    -   residues 50-66 of SEQ ID NO:172 (CDR-H2 VH. 1a 1A11);    -   residues 50-66 of SEQ ID NO:173 (CDR-H2 VH.1b 1A11);    -   residues 50-66 of SEQ ID NO:174 (CDR-H2 VH.2a 1A11);    -   residues 50-66 of SEQ ID NO:179 (CDR-H2 VH.1 38H12);    -   residues 50-66 of SEQ ID NO:180 (CDR-H2 VH.1A 38H12);    -   residues 50-66 of SEQ ID NO:181 (CDR-H2 VH.1b 38H12);    -   residues 31-35 of SEQ ID NO:182 (CDR-H1 VH.2a 38H12);    -   residues 50-66 of SEQ ID NO:187 (CDR-H2 h1A11VH.1);    -   residues 50-66 of SEQ ID NO:188 (CDR-H2 h1A11.A6);    -   residues 50-66 of SEQ ID NO:189 (CDR-H2 h1A11.A8);    -   residues 50-66 of SEQ ID NO:190 (CDR-H2 h1A11.C6);    -   residues 50-66 of SEQ ID NO:191 (CDR-H2 h1A11.A11);    -   residues 50-66 of SEQ ID NO:192 (CDR-H2 h1A11.B5);    -   residues 50-66 of SEQ ID NO:193 (CDR-H2 h1A11.E12);    -   residues 50-66 of SEQ ID NO:194 (CDR-H2 h1A11.G3);    -   residues 50-66 of SEQ ID NO:195 (CDR-H2 h1A11.F5); and    -   residues 50-66 of SEQ ID NO:196 (CDR-H2 h1A11.H2);

CDR-H3 is selected from the group consisting of:

-   -   X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—X₉ (SEQ ID NO:153), wherein;        -   X₁ is G;        -   X₂ is Y;        -   X₃ is Y;        -   X₄ is N;        -   X₅ is S;        -   X₆ is P;        -   X₇ is F;        -   X₈ is A; and        -   X₉ is Y, F, or S;    -   residues 99-107 of SEQ ID NO:157 (CDR-H3 38H12);    -   residues 101-111 of SEQ ID NO:161 (CDR-H3 37D10);    -   residues 99-105 of SEQ ID NO:163 (CDR-H3 32C7);    -   residues 99-105 of SEQ ID NO:165 (CDR-H3 14G1);    -   residues 99-110 of SEQ ID NO:167 (CDR-H3 14A11);    -   residues 99-110 of SEQ ID NO:169 (CDR-H3 15D6);    -   residues 99-107 of SEQ ID NO:171 (CDR-H3 VH.1 1A11);    -   residues 99-107 of SEQ ID NO:172 (CDR-H3 VH.1a 1A11);    -   residues 99-107 of SEQ ID NO:173 (CDR-H3 VH.1b 1A11);    -   residues 99-107 of SEQ ID NO:174 (CDR-H3 VH.2a 1A11);    -   residues 99-107 of SEQ ID NO:179 (CDR-H3 VH.1 38H12);    -   residues 99-107 of SEQ ID NO:180 (CDR-H3 VH.1A 38H12);    -   residues 99-107 of SEQ ID NO:181 (CDR-H2 VH.1b 38H12);    -   residues 99-107 of SEQ ID NO:182 (CDR-H1 VH.2a 38H12);    -   residues 99-107 of SEQ ID NO:187 (CDR-H3 h1A11VH.1);    -   residues 99-107 of SEQ ID NO:188 (CDR-H3 h1A11.A6);    -   residues 99-107 of SEQ ID NO:189 (CDR-H3 h1A11.A8);    -   residues 99-107 of SEQ ID NO:190 (CDR-H3 h1A11.C6);    -   residues 99-107 of SEQ ID NO:191 (CDR-H3 h1A11.A11);    -   residues 99-107 of SEQ ID NO:192 (CDR-H3 h1A11.B5);    -   residues 99-107 of SEQ ID NO:193 (CDR-H3 h1A11.E12);    -   residues 99-107 of SEQ ID NO:194 (CDR-H3 h1A11.G3);    -   residues 99-107 of SEQ ID NO:195 (CDR-H3 h1A11.F5); and    -   residues 99-107 of SEQ ID NO:196 (CDR-H3 h1A11.H2);

CDR-L1 is selected from the group consisting of:

-   -   X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—X₉—X₁₀—X₁₁ (SEQ ID NO:154), wherein;        -   X₁ is R;        -   X₂ is A;        -   X₃ is S;        -   X₄ is E or Q;        -   X₅ is D or E;        -   X₆ is I;        -   X₇ is Y or W;        -   X₈ is S, I, Y, N, or R;        -   X₉ is N;        -   X₁₀ is L; and        -   X₁₁ is A;    -   residues 24-34 of SEQ ID NO:158 (CDR-L1 38H12);    -   residues 24-34 of SEQ ID NO:162 (CDR-L1 37D10);    -   residues 24-34 of SEQ ID NO:164 (CDR-L1 32C7);    -   residues 24-34 of SEQ ID NO:166 (CDR-L1 14G1);    -   residues 23-37 of SEQ ID NO:168 (CDR-L1 14A11);    -   residues 23-37 of SEQ ID NO:170 (CDR-L1 15D6);    -   residues 24-34 of SEQ ID NO:175 (CDR-L1 VL.1 1A11);    -   residues 24-34 of SEQ ID NO:176 (CDR-L1 VL.1a 1A11);    -   residues 24-34 of SEQ ID NO:177 (CDR-L1 VL.1b 1A11);    -   residues 24-34 of SEQ ID NO:178 (CDR-L1 VL.2a 1A11);    -   residues 24-34 of SEQ ID NO:183 (CDR-L1 VL.1 38H12);    -   residues 24-34 of SEQ ID NO:184 (CDR-L1 VL.1a 38H12);    -   residues 24-34 of SEQ ID NO:185 (CDR-L1 VL.1b 38H12);    -   residues 24-34 of SEQ ID NO:186 (CDR-L1 VL.2a 38H12);    -   residues 24-34 of SEQ ID NO:197 (CDR-L1 h1A11VL.1);    -   residues 24-34 of SEQ ID NO:198 (CDR-L1 h1A11.A2);    -   residues 24-34 of SEQ ID NO:199 (CDR-L1 h1A11.A12);    -   residues 24-34 of SEQ ID NO:200 (CDR-L1 h1A11.A7);    -   residues 24-34 of SEQ ID NO:201 (CDR-L1 h1A11.B4);    -   residues 24-34 of SEQ ID NO:202 (CDR-L1 h1A11.B5); and    -   residues 24-34 of SEQ ID NO:203 (CDR-L1 h1A11.E12);

CDR-L2 is selected from group consisting of:

-   -   X₁—X₂—X₃—X₄—X₅—X₆—X₇ (SEQ ID NO:155), wherein;        -   X₁ is D;        -   X₂ is T;        -   X₃ is N or S;        -   X₄ is N, D, S, I, Y, or V;        -   X₅ is L;        -   X₆ is A; and        -   X₇ is D;    -   residues 50-56 of SEQ ID NO:158 (CDR-L2 38H12);    -   residues 50-56 of SEQ ID NO:162 (CDR-L2 37D10);    -   residues 50-56 of SEQ ID NO:164 (CDR-L2 32C7);    -   residues 50-56 of SEQ ID NO:166 (CDR-L2 14G1);    -   residues 53-59 of SEQ ID NO:168 (CDR-L2 14A11);    -   residues 53-59 of SEQ ID NO:170 (CDR-L2 15D6);    -   residues 50-56 of SEQ ID NO:175 (CDR-L2 VL.1 1A11);    -   residues 50-56 of SEQ ID NO:176 (CDR-L2 VL.1a 1A11);    -   residues 50-56 of SEQ ID NO:177 (CDR-L2 VL.1b 1A11);    -   residues 50-56 of SEQ ID NO:178 (CDR-L2 VL.2a 1A11);    -   residues 50-56 of SEQ ID NO:183 (CDR-L2 VL.1 38H12);    -   residues 50-56 of SEQ ID NO:184 (CDR-L2 VL.1a 38H12);    -   residues 50-56 of SEQ ID NO:185 (CDR-L2 VL.1b 38H12);    -   residues 50-56 of SEQ ID NO:186 (CDR-L2 VL.2a 38H12);    -   residues 50-56 of SEQ ID NO:197 (CDR-L2 h1A11VL.1);    -   residues 50-56 of SEQ ID NO:198 (CDR-L2 h1A11.A2);    -   residues 50-56 of SEQ ID NO:199 (CDR-L2 h1A11.A12);    -   residues 50-56 of SEQ ID NO:200 (CDR-L2 h1A11.A7);    -   residues 50-56 of SEQ ID NO:201 (CDR-L2 h1A11.B4);    -   residues 50-56 of SEQ ID NO:202 (CDR-L2 h1A11.B5); and    -   residues 50-56 of SEQ ID NO:203 (CDR-L2 h1A11.E12); and

CDR-L3 is selected from the group consisting of:

-   -   X₁—X₂—X₃—X₄—X₅—X₆—X₇—X₈—X₉ (SEQ ID NO:156), wherein;        -   X₁ is Q;        -   X₂ is Q;        -   X₃ is Y;        -   X₄ is N, D, or T;        -   X₅ is N, Y, or W;        -   X₆ is Y or V;        -   X₇ is P;        -   X₈ is P; and        -   X₉ is T;    -   residues 89-97 of SEQ ID NO:158 (CDR-L3 38H12);    -   residues 89-97 of SEQ ID NO:162 (CDR-L3 37D10);    -   residues 89-97 of SEQ ID NO:164 (CDR-L3 32C7);    -   residues 89-98 of SEQ ID NO:166 (CDR-L3 14G1);    -   residues 92-100 of SEQ ID NO:168 (CDR-L3 14A11);    -   residues 92-100 of SEQ ID NO:170 (CDR-L3 15D6);    -   residues 89-97 of SEQ ID NO:175 (CDR-L3 VL.1 1A11);    -   residues 89-97 of SEQ ID NO:176 (CDR-L3 VL.1a 1A11);    -   residues 89-97 of SEQ ID NO:177 (CDR-L3 VL.1b 1A11);    -   residues 89-97 of SEQ ID NO:178 (CDR-L3 VL.2a 1A11);    -   residues 89-97 of SEQ ID NO:183 (CDR-L3 VL.1 38H12);    -   residues 89-97 of SEQ ID NO:184 (CDR-L3 VL.1a 38H12);    -   residues 89-97 of SEQ ID NO:185 (CDR-L3 VL.1b 38H12);    -   residues 89-97 of SEQ ID NO:186 (CDR-L3 VL.2a 38H12);    -   residues 89-97 of SEQ ID NO:197 (CDR-L3 h1A11VL.1);    -   residues 89-97 of SEQ ID NO:198 (CDR-L3 h1A11.A2);    -   residues 89-97 of SEQ ID NO:199 (CDR-L3 h1A11.A12);    -   residues 89-97 of SEQ ID NO:200 (CDR-L3 h1A11.A7);    -   residues 89-97 of SEQ ID NO:201 (CDR-L3 h1A11.B4);    -   residues 89-97 of SEQ ID NO:202 (CDR-L3 h1A11.B5); and    -   residues 89-97 of SEQ ID NO:203 (CDR-L3 h1A11.E12).

Preferably, a DLL4 binding protein comprises at least one CDR describedabove, more preferably any two CDRs described above, more preferably anythree CDRs described above, even more preferably any four CDRs describedabove, still more preferably any five CDRs described above, and mostpreferably any six CDRs described above (i.e., CDR-H1, CDR-H2, CDR-H3,CDR-L1, CDR-L2, and CDR-L3 as described above). A particularly preferredDLL4 binding protein comprising three CDRs comprises CDR-H1, CDR-H2, andCDR-H3 as described above.

Preferably, a DLL4 binding protein comprising one or more CDRs describedabove binds human (“hu”, “h”) DLL4 and also one or more DLL4 proteinsselected from the group consisting of: mouse (“murine”, “mu”) DLL4,cynomolgus monkey (“cynomolgus”, “cyno”) DLL4, and rat DLL4.

Preferably, a DLL4 binding protein comprising one or more CDRs describedabove binds human (“hu”) DLL4 and also cynomolgus monkey (“cynomolgus”,“cyno”) DLL4.

2. Anti-DLL4 Chimeric Antibodies.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different animal species, such as antibodieshaving a variable region derived from a murine monoclonal antibody and ahuman immunoglobulin constant region. See e.g., Morrison, Science, 229:1202-1207 (1985); Oi et al., BioTechniques, 4: 214 (1986); Gillies etal., J. Immunol. Methods, 125: 191-202 (1989); U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816,397. In addition, techniques developed for theproduction of “chimeric antibodies” by splicing genes from a mouseantibody molecule of appropriate antigen specificity together with genesfrom a human antibody molecule of appropriate biological activity can beused. See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Neuberger et al., Nature, 312: 604-608 (1984); Takedaet al., Nature, 314: 452-454 (1985), which are incorporated herein byreference in their entireties.

3. Anti-DLL4 CDR Grafted Antibodies.

The isolated anti-DLL4 antibody CDR sequences of the invention may beused to make CDR-grafted antibodies to modulate the properties of theoriginal antibody. Such properties include but are not limited tobinding kinetics, affinity, biological activities, speciescross-reactivity, molecule cross-reactivity, epitope, physicochemicalproperties, pharmacokinetic properties, pharmacodynamic properties, orpharmacological properties. CDR-grafted antibodies comprise heavy andlight chain variable region sequences from a human antibody or anon-human primate antibody wherein one or more of the CDR regions of VHand/or VL are replaced with CDR sequences of the original anti-DLL4antibody. A framework sequence from any human or non-human primateantibody may serve as the template for CDR grafting. However, straightchain replacement onto such a framework often leads to some loss ofbinding affinity to the antigen. The more homologous a human, or otherspecies, antibody is to the original human antibody, the less likely thepossibility that combining the CDRs with the new human framework ornon-human primate framework will introduce distortions in the CDRs thatcould reduce affinity or other properties. Therefore, it is preferablethat the variable framework that is chosen to replace the human variableregion framework apart from the CDRs has at least a 30% sequenceidentity with the human antibody variable region framework. It is morepreferable that the variable region framework that is chosen to replacethe human variable region framework apart from the CDRs has at least a40% sequence identity with the human antibody variable region framework.It is more preferable that the variable region framework that is chosento replace the human variable framework apart from the CDRs has at leasta 50% sequence identity with the human antibody variable regionframework. It is more preferable that the variable region framework thatis chosen to replace the human variable framework apart from the CDRshas at least a 60% sequence identity with the human antibody variableregion framework. It is more preferable that the new human or non-humanprimate and the original human variable region framework apart from theCDRs has at least 70% sequence identity. It is even more preferable thatthe new human or non-human primate and the original human variableregion framework apart from the CDRs has at least 75% sequence identity.It is most preferable that the new human or non-human primate and theoriginal human variable region framework apart from the CDRs has atleast 80% sequence identity. Even using a highly homologous human ornon-human primate framework to graft CDRs of the original humananti-DLL4 antibody, the resulting grafted antibody may still losebinding affinity to antigen to some degree. In this case, to regain theaffinity it is necessary to include at least one or more key frameworkresidue(s) substitution of the original antibody to the correspondingposition of the newly grafted antibody. Such a key residue may beselected from the group consisting of:

-   -   a residue adjacent to a CDR;    -   a glycosylation site residue;    -   a rare residue;    -   a residue capable of interacting with human DLL4    -   a canonical residue;    -   a contact residue between heavy chain variable region and light        chain variable region;    -   a residue within a Vernier zone; and    -   a residue in a region that overlaps between a Chothia-defined        variable heavy chain CDR1 and a Kabat-defined first heavy chain        framework.

4. Anti-DLL4 Humanized Antibodies.

While the compositions of the present invention eliminate therequirement to make humanized antibodies, humanized DLL4 antibodies maybe prepared using compositions of the invention. Humanized antibodiesare antibody molecules from non-human species antibody that binds thedesired antigen having one or more complementarity determining regions(CDRs) from the non-human species and framework regions from a humanimmunoglobulin molecule. Known human Ig sequences are disclosed at websites available via the world wide web (www.), e.g.,ncbi.nlm.nih.gov/entrez/query.fcgi; atcc.org/phage/hdb.html;sciquest.com/; abcam.com/; antibodyresource.com/onlinecomp.html;public.iastate.edu/.about.pedro-/research_tools.html;mgen.uniheidelberg.de/SD/IT/IT.html;whfreeman.com/immunology-/CH05/kuby05.htm;library.thinkquest.org/12429/Immune/Antibody.html;hhmi.org/grants/lectures/1996/vlab/;path.-cam.ac.uk/.about.mrc7/mikeimages.html; antibodyresource.com/;mcb.harvard.edu/BioLinks-/Immunology.html; immunologylink.com/;pathbox.wustl.edu/about.hcenter/index.html; bio-tech.ufl.edu/about.hcl/;pebio.com/pa/340913-/340913.html; nal.usda.gov/awic/pubs/antibody/;m.ehimeu.acjp/.about.yasuhito-/Elisa.html; biodesign.com/table.asp;icnet.uk/axp/facs/davies/lin-ks.html;biotech.ufl.edu-/.about.fccl/protocol.html; isac-net.org/sites_geo.html;aximtl.imt.uni-marburg.de/.about.rek/AEP-Start.html;baserv.uci.kun.nl/.about.jraats/linksl.html;recab.uni-hd.de/immuno.bme.nwu.edu/;mrc-cpe.cam.ac.uk/imt-doc/public/INTRO.html;ibt.unam.mx/-vir/V_mice.html; imgt.cnusc.fr: 8104/;biochem.ucl.ac.uk/.about.martin/abs/index.html; anti-body.bath.ac.uk/;abgen.cvm.tamu.edu/lab/wwwabgen.html;unizh.ch/.about.honegger/AHO-seminar/Slide01.html;cryst.bbk.ac.uk/.about.ubcg07s/; nimr.mrc.ac.uk/CC/ccaewg/ccaewg.htm;path.cam.ac.uk/.about.mrc7/humanisation/TAHHP.html;ibt.unam.mx/vir/structure/stat_aim.-html;biosci.missouri.edu/smithgp/index.html;cryst.bioc.cam.ac.uk/.about.fmolina/Webpages-/Pept/spottech.html;jerini.de/frroducts.htm; patents.ibm.com/ibm.html. Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Dept. Health(1983), each entirely incorporated herein by reference. Such importedsequences can be used to reduce immunogenicity or reduce, enhance ormodify binding, affinity, on-rate, off-rate, avidity, specificity,half-life, or any other suitable characteristic, as known in the art.

Framework residues in the human framework regions may be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,U.S. Pat. No. 5,585,089 (Queen et al.); Riechmann et al., Nature, 332:323-327 (1988), which are incorporated herein by reference in theirentireties.) Three-dimensional immunoglobulin models are commonlyavailable and are familiar to those skilled in the art. Computerprograms are available which illustrate and display probablethree-dimensional conformational structures of selected candidateimmunoglobulin sequences. Inspection of these displays permits analysisof the likely role of the residues in the functioning of the candidateimmunoglobulin sequence, i.e., the analysis of residues that influencethe ability of the candidate immunoglobulin to bind its antigen. In thisway, FR residues can be selected and combined from the consensus andimport sequences so that the desired antibody characteristic, such asincreased affinity for the target antigen(s), is achieved. In general,the CDR residues are directly and most substantially involved ininfluencing antigen binding. Antibodies can be humanized using a varietyof techniques known in the art, such as but not limited to thosedescribed in Jones et al., Nature, 321: 522-525 (1986); Verhoeyen etal., Science, 239: 1534-1536 (1988), Sims et al., J. Immunol., 151:2296-2308 (1993); Chothia and Lesk, J. Mol. Biol., 196: 901-917 (1987),Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285-4289 (1992); Prestaet al., J. Immunol., 151: 2623-2632 (1993), Padlan, E. A., MolecularImmunology, 28(4/5): 489-498 (1991); Studnicka et al., ProteinEngineering, 7(6): 805-814 (1994); Roguska. et al., Proc. Natl. Acad.Sci. USA, 91:969-973 (1994); PCT Publication Nos. WO 91/09967, WO99/06834 (PCT/US98/16280), WO 97/20032 (PCT/US96/18978), WO 92/11272(PCT/US91/09630), WO 92/03461 (PCT/US91/05939), WO 94/18219(PCT/US94/01234), WO 92/01047 (PCT/GB91/01134), WO 93/06213(PCT/GB92/01755), WO90/14443, WO90/14424, and WO90/14430; EuropeanPublication Nos. EP 0 592 106, EP 0 519 596, and EP 0 239 400; U.S. Pat.Nos. 5,565,332; 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476;5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370;5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567, each entirelyincorporated herein by reference, included references cited therein.

C. Production of Antibodies and Antibody-Producing Cell Lines.

Preferably, anti-DLL4 antibodies of the present invention exhibit a highcapacity to reduce or to neutralize tumor angiogenesis activity, e.g.,as assessed by any one of several in vitro and in vivo assays known inthe art. For example, these antibodies neutralize DLL4 interaction inthe Notch-signaling pathway with IC₅₀ values in DLL4 in the range of atleast about 10⁻⁷M, or about 10⁻⁸ M. Preferably, anti-DLL4 antibodies ofthe present invention also exhibit a high capacity to reduce or toneutralize DLL4 activity.

In preferred embodiments, an isolated antibody, or antigen-bindingportion thereof, binds human DLL4, wherein the antibody, orantigen-binding portion thereof, dissociates from human DLL4 with aK_(off) rate constant of about 0.1 s⁻¹ or less, as determined by surfaceplasmon resonance, or which inhibits DLL4 and/or human DLL4 activitywith an IC₅₀ of about 1×10⁻⁶ M or less. Alternatively, the antibody, oran antigen-binding portion thereof, may dissociate from human DLL4 witha K_(off) rate constant of about 1×10⁻² s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit human DLL4 and/or human DLL4activity with an IC₅₀ of about 1×10⁻⁷ M or less. Alternatively, theantibody, or an antigen-binding portion thereof, may dissociate fromhuman DLL4 with a K_(off) rate constant of about 1×10⁻³ s⁻¹ or less, asdetermined by surface plasmon resonance, or may inhibit human DLL4 withan IC₅₀ of about 1×10⁻⁸ M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from human DLL4 with aK_(off) rate constant of about 1×10⁻⁴ s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit DLL4 activity with an IC₅₀ ofabout 1×10⁻⁹M or less. Alternatively, the antibody, or anantigen-binding portion thereof, may dissociate from human DLL4 with aK_(off) rate constant of about 1×10⁻⁵s⁻¹ or less, as determined bysurface plasmon resonance, or may inhibit DLL4 and/or human DLL4activity with an IC₅₀ of about 1×10⁻¹⁰ M or less. Alternatively, theantibody, or an antigen-binding portion thereof, may dissociate fromhuman DLL4 with a K_(off) rate constant of about 1×10⁻⁵s⁻¹ or less, asdetermined by surface plasmon resonance, or may inhibit DLL4 and/orhuman DLL4 activity with an IC₅₀ of about 1×10⁻¹¹M or less.

In certain embodiments, the antibody comprises a heavy chain constantregion, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgDconstant region. Preferably, the heavy chain constant region is an IgG1heavy chain constant region or an IgG4 heavy chain constant region.Furthermore, the antibody can comprise a light chain constant region,either a kappa light chain constant region or a lambda light chainconstant region. Preferably, the antibody comprises a kappa light chainconstant region. Alternatively, the antibody portion can be, forexample, a Fab fragment or a single chain Fv fragment.

Replacements of amino acid residues in the Fc portion to alter antibodyeffector function are known in the art (see, U.S. Pat. Nos. 5,648,260and 5,624,821 (Winter et al.)). The Fc portion of an antibody mediatesseveral important effector functions e.g. cytokine induction, ADCC,phagocytosis, complement dependent cytotoxicity (CDC), andhalf-life/clearance rate of antibody and antigen-antibody complexes. Insome cases these effector functions are desirable for therapeuticantibody but in other cases might be unnecessary or even deleterious,depending on the therapeutic objectives. Certain human IgG isotypes,particularly IgG1 and IgG3, mediate ADCC and CDC via binding to FcγRsand complement C1q, respectively. Neonatal Fc receptors (FcRn) are thecritical components determining the circulating half-life of antibodies.In still another embodiment at least one amino acid residue is replacedin the constant region of the antibody, for example the Fc region of theantibody, such that effector functions of the antibody are altered.

One embodiment provides a labeled binding protein wherein an antibody orantibody portion of the invention is derivatized or linked to anotherfunctional molecule (e.g., another peptide or protein). For example, alabeled binding protein of the invention can be derived by functionallylinking an antibody or antibody portion of the invention (by chemicalcoupling, genetic fusion, noncovalent association or otherwise) to oneor more other molecular entities, such as another antibody (e.g., abispecific antibody or a diabody), a detectable agent, a cytotoxicagent, a pharmaceutical agent, and/or a protein or peptide that canmediate associate of the antibody or antibody portion with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

Useful detectable agents with which an antibody or antibody portion ofthe invention may be derivatized include fluorescent compounds.Exemplary fluorescent detectable agents include fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin and the like. An antibody may also bederivatized with detectable enzymes, such as alkaline phosphatase,horseradish peroxidase, glucose oxidase and the like. When an antibodyis derivatized with a detectable enzyme, it is detected by addingadditional reagents that the enzyme uses to produce a detectablereaction product. For example, when the detectable agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which isdetectable. An antibody may also be derivatized with biotin, anddetected through indirect measurement of avidin or streptavidin binding.

Another embodiment of the invention provides a crystallized DLL4 bindingprotein. Preferably, the invention relates to crystals of DLL4 bindingproteins described herein, including whole anti-DLL4 antibodies,fragments thereof, as well as antibody constructs and binding proteinconjugates (including antibody conjugates) as disclosed herein, andformulations and compositions comprising such crystals. In oneembodiment, the crystallized binding protein has a greater half-life invivo than the soluble counterpart of the binding protein. In anotherembodiment the binding protein retains biological activity aftercrystallization. Crystallized binding proteins of the invention may beproduced according methods known in the art and as disclosed in PCTPublication No. WO 02/72636, incorporated herein by reference.

Another embodiment of the invention provides a glycosylated bindingprotein wherein the antibody or antigen-binding portion thereofcomprises one or more carbohydrate residues. Nascent in vivo proteinproduction may undergofurther processing, known as post-translationalmodification. In particular, sugar (glycosyl) residues may be addedenzymatically, a process known as glycosylation. The resulting proteinsbearing covalently linked oligosaccharide side chains are known asglycosylated proteins or glycoproteins. Protein glycosylation depends onthe amino acid sequence of the protein of interest, as well as the hostcell in which the protein is expressed. Different organisms may producedifferent glycosylation enzymes (e.g., glycosyltransferases andglycosidases), and have different substrates (nucleotide sugars)available. Due to such factors, protein glycosylation pattern, andcomposition of glycosyl residues, may differ depending on the hostsystem in which the particular protein is expressed. Glycosyl residuesuseful in the invention may include, but are not limited to, glucose,galactose, mannose, fucose, n-acetylglucosamine and sialic acid.Preferably the glycosylated binding protein comprises glycosyl residuessuch that the glycosylation pattern is human.

It is known to those skilled in the art that differing proteinglycosylation may result in differing protein characteristics. Forinstance, the efficacy of a therapeutic protein produced in amicroorganism host, such as yeast, and glycosylated utilizing the yeastendogenous pathway may be reduced compared to that of the same proteinexpressed in a mammalian cell, such as a CHO cell line. Suchglycoproteins may also be immunogenic in humans and show reducedhalf-life in vivo after administration. Specific receptors in humans andother animals may recognize specific glycosyl residues and promote therapid clearance of the protein from the bloodstream. Other adverseeffects may include changes in protein folding, solubility,susceptibility to proteases, trafficking, transport,compartmentalization, secretion, recognition by other proteins orfactors, antigenicity, or allergenicity. Accordingly, a practitioner mayprefer a therapeutic protein with a specific composition and pattern ofglycosylation, for example glycosylation composition and patternidentical, or at least similar, to that produced in human cells or inthe species-specific cells of the intended subject animal.

Expressing glycosylated proteins different from that of a host cell maybe achieved by genetically modifying the host cell to expressheterologous glycosylation enzymes. Using techniques known in the art apractitioner may generate antibodies or antigen-binding portions thereofexhibiting human protein glycosylation. For example, yeast strains havebeen genetically modified to express non-naturally occurringglycosylation enzymes such that glycosylated proteins (glycoproteins)produced in these yeast strains exhibit protein glycosylation identicalto that of animal cells, especially human cells (US Patent ApplicationPublication Nos. 2004/0018590 and 2002/0137134).

Further, it will be appreciated by one skilled in the art that a proteinof interest may be expressed using a library of host cells geneticallyengineered to express various glycosylation enzymes, such that memberhost cells of the library produce the protein of interest with variantglycosylation patterns. A practitioner may then select and isolate theprotein of interest with particular novel glycosylation patterns.Preferably, the protein having a particularly selected novelglycosylation pattern exhibits improved or altered biologicalproperties.

D. Uses of DLL4 Binding Proteins.

Given their ability to bind to human DLL4 and murine DLL4, the DLL4binding proteins described herein, including antibodies and portionsthereof, can be used to detect or measure DLL4 in a sample (e.g., in amixture, solution, or biological sample, such as blood, serum, orplasma), using any of the conventional immunoassays known in the art,such as an enzyme linked immunosorbent assays (ELISA), aradioimmunoassay (RIA), or a tissue immunohistochemistry. The inventionprovides a method for detecting human DLL4 and/or murine DLL4 in asample comprising contacting a sample with a DLL4 binding protein anddetecting either the DLL4 binding protein bound to human DLL4 and/ormurine DLL4 or the unbound binding protein to thereby detect human DLL4and/or murine DLL4 in the sample. A DLL4 binding protein describedherein can be directly or indirectly labeled with a detectable substancetofacilitate detection of the bound or unbound DLL4 binding protein.Suitable detectable substances include various enzymes, prostheticgroups, fluorescent materials, luminescent materials and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; and examples ofsuitable radioactive material include ³H, ¹⁴C, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I, ¹⁷⁷Lu, ¹⁶⁶Ho, or ¹⁵³Sm.

Biological samples that can be assayed for DLL4 include urine, feces,blood, serum, plasma, perspiration, saliva, oral swab (cheek, tongue,throat), vaginal swab, rectal swab, dermal swab, dermal scrape, tissuebiopsy, as well as any other tissue sample that can be obtained bymethods available in the art.

Alternative to labeling the binding protein, human DLL4 can be assayedin biological fluids by a competition immunoassay utilizing recombinanthuman (rh) DLL4 standards labeled with a detectable substance and anunlabeled DLL4 binding protein described herein. In this assay, thebiological sample, the labeled rhDLL4 standards, and the DLL4 bindingprotein are combined and the amount of labeled rhDLL4 standard bound tothe unlabeled binding protein is determined. The amount of human DLL4 inthe biological sample is inversely proportional to the amount of labeledrhDLL4 standard bound to the DLL4 binding protein. Similarly, human DLL4can also be assayed in biological fluids by a competition immunoassayutilizing rhDLL4 standards labeled with a detectable substance and anunlabeled DLL4 binding protein described herein.

The DLL4 binding proteins of the invention preferably are capable ofneutralizing DLL4 activity, in particular hDLL4 activity, both in vitroand in vivo. Accordingly, such binding proteins of the invention can beused to inhibit DLL4 activity, e.g., in a cell culture containing DLL4,in human subjects, or in other mammalian subjects expressing a DLL4 withwhich a binding protein of the invention cross-reacts. In oneembodiment, the invention provides a method for inhibiting DLL4 activitycomprising contacting a DLL4 with a DLL4 antibody or antibody portion ofthe invention such that DLL4 activity is inhibited. For example, in acell culture containing or suspected of containing DLL4, an antibody orantibody portion of the invention can be added to the culture medium toinhibit DLL4 activity in the culture.

In another embodiment, the invention provides a method for reducing DLL4activity in a subject, advantageously from a subject suffering from adisease or disorder in which DLL4 or DLL4 activity is detrimental. Theinvention provides methods for reducing DLL4 or DLL4 activity in asubject suffering from such a disease or disorder, which methodcomprises administering to the subject a DLL4 binding protein of theinvention such that DLL4 or DLL4 activity in the subject is reduced.Preferably, the DLL4 is human DLL4, and the subject is a human subject.Alternatively, the subject can be a mammal expressing a DLL4 to which aDLL4 binding protein of the invention is capable of binding. Stillfurther, the subject can be a mammal into which DLL4 has been introduced(e.g., by administration of DLL4 or by expression of a DLL4 transgene).An antibody or other DLL4 binding protein of the invention can beadministered to a human subject for therapeutic purposes. Moreover, aDLL4 binding protein of the invention can be administered to a non-humanmammal expressing a DLL4 with which the binding protein is capable ofbinding for veterinary purposes or as an animal model of human disease.Regarding the latter, such animal models may be useful for evaluatingthe therapeutic efficacy of antibodies and other DLL4 binding proteinsof the invention (e.g., testing of dosages and time courses ofadministration).

As used herein, the term “a disorder in which DLL4 and/or Notchsignaling activity is detrimental” is intended to include diseases, suchas cancer, and other disorders in which the presence of DLL4 and/orNotch signaling activity in a subject suffering from the disorder hasbeen shown to be or is suspected of being either responsible for thepathophysiology of the disorder or a factor that contributes to aworsening of the disorder. Accordingly, a disorder in which DLL4 and/orNotch signaling activity is detrimental is a disorder in which reductionof DLL4 and/or Notch signaling activity is expected to alleviate thesymptoms and/or progression of the disorder (e.g., tumor growth). Suchdisorders may be evidenced, for example, by an increase in angiogenesisin a subject suffering from the disorder (e.g., an increase in theconcentration of various proteins known in the art to increase in serum,plasma, synovial fluid, etc., of the subject during tumor growth andformation), which can be detected, for example, using an anti-DLL4antibody as described above. Non-limiting examples of disorders that canbe treated with the antibodies of the invention include those disordersdiscussed in the section below pertaining to pharmaceutical compositionsof the antibodies of the invention.

II. Pharmaceutical Compositions and Therapeutic Uses.

The invention also provides pharmaceutical compositions comprising aDLL4 binding protein of the invention and a pharmaceutically acceptablecarrier. The pharmaceutical compositions comprising DLL4 bindingproteins of the invention are for use in, but not limited to,diagnosing, detecting, or monitoring a disorder; in preventing,treating, managing, or ameliorating a disorder or one or more symptomsthereof; and/or in research. In a specific embodiment, a compositioncomprises one or more DLL4 binding proteins of the invention. In anotherembodiment, the pharmaceutical composition comprises one or more bindingproteins of the invention and one or more prophylactic or therapeuticagents other than binding proteins of the invention for treating adisorder in which DLL4 and/or DLL4 activity is detrimental. Preferably,the prophylactic or therapeutic agents known to be useful for or havingbeen or currently being used in the prevention, treatment, management,or amelioration of a disorder, such as cancer or a tumor, or one or moresymptoms thereof. In accordance with these embodiments, the compositionmay further comprise of a carrier, diluent, or excipient.

The binding proteins of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject.Typically, the pharmaceutical composition comprises a DLL4 bindingprotein (or DLL4 binding portion thereof) of the invention and apharmaceutically acceptable carrier. As used herein, “pharmaceuticallyacceptable carrier” includes any and all solvents, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like that are physiologically compatible.Examples of pharmaceutically acceptable carriers include one or more ofwater, saline, phosphate buffered saline, dextrose, glycerol, ethanoland the like, as well as combinations thereof. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.Pharmaceutically acceptable carriers may further comprise minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the antibody or antibody portion.

Various delivery systems are known and can be used to administer one ormore DLL4 binding proteins of the invention or the combination of one ormore binding proteins of the invention and a prophylactic agent ortherapeutic agent useful for preventing, managing, treating, orameliorating a disorder or one or more symptoms thereof, e.g., reducingtumor angiogenesis, encapsulation in liposomes, microparticles,microcapsules, recombinant cells capable of expressing the DLL4 bindingprotein, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol.Chem., 262: 4429-4432 (1987)), construction of a nucleic acid as part ofa retroviral or other vector, etc. Methods of administering aprophylactic or therapeutic agent of the invention include, but are notlimited to, parenteral administration (e.g., intradermal, intramuscular,intraperitoneal, intravenous and subcutaneous), epidural administration,intratumoral administration, and mucosal administration (e.g.,intranasal and oral routes). In addition, pulmonary administration canbe employed, e.g., by use of an inhaler or nebulizer, and formulationwith an aerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968;5,985,320; 5,985,309; 5,934, 272; 5,874,064; 5,855,913; 5,290,540; and4,880,078; and PCT Publication Nos. WO 92/19244, WO 97/32572, WO97/44013, WO 98/31346, and WO 99/66903, each of which is incorporatedherein by reference their entireties. In one embodiment, a DLL4 bindingprotein of the invention, combination therapy, or a composition of theinvention is administered using Alkermes AIR® pulmonary drug deliverytechnology (Alkermes, Inc., Cambridge, Mass., US). In a specificembodiment, prophylactic or therapeutic agents of the invention areadministered intramuscularly, intravenously, intratumorally, orally,intranasally, pulmonary, or subcutaneously. The prophylactic ortherapeutic agents may be administered by any convenient route, forexample by infusion or bolus injection, by absorption through epithelialor mucocutaneous linings (e.g., oral mucosa, rectal and intestinalmucosa, etc.) and may be administered together with other biologicallyactive agents. Administration can be systemic or local.

In a specific embodiment, it may be desirable to administer theprophylactic or therapeutic agents of the invention locally to the areain need of treatment; this may be achieved by, for example, and not byway of limitation, local infusion, by injection, or by means of animplant, said implant being of a porous or non-porous material,including membranes and matrices, such as sialastic membranes, polymers,fibrous matrices (e.g., Tissuel®), or collagen matrices. In oneembodiment, an effective amount of one or more DLL4 binding proteins ofthe invention antagonists is administered locally to the affected areato a subject to prevent, treat, manage, and/or ameliorate a disorder ora symptom thereof. In another embodiment, an effective amount of one ormore DLL4 binding proteins of the invention is administered locally tothe affected area in combination with an effective amount of one or moretherapies (e.g., one or more prophylactic or therapeutic agents) otherthan a binding protein of the invention of a subject to prevent, treat,manage, and/or ameliorate a disorder or one or more symptoms thereof.

In another embodiment, the prophylactic or therapeutic agent can bedelivered in a controlled release or sustained release system. In oneembodiment, a pump may be used to achieve controlled or sustainedrelease (see, Langer (Science, 249: 1527-1533 (1990)); Sefton, CRC Crit.Ref. Biomed. Eng., 14: 201-240 (1987); Buchwald et al., Surgery, 88:507-516 (1980); Saudek et al., N. Engl. J. Med., 321: 574-579 (1989)).In another embodiment, polymeric materials can be used to achievecontrolled or sustained release of the therapies of the invention. See,e.g., Goodson, J. M, In Medical Applications of Controlled Release, Vol.II, Applications and Evaluations, (Langer and Wise, eds.), (CRC PressInc., Boca Raton, 1984), chapter 6, pages 115-138; Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.) (Wiley, New York, 1984); Langer and Peppas, J. Macromol. Sci.Rev. Macromol. Chem. Phys., C23: 61-126 (1983); see also, Levy et al.,Science, 228: 190-192 (1985); During et al., Ann. Neurol., 25: 351-356(1989); Howard et al., J. Neurosurg., 71: 105-112 (1989); U.S. Pat. Nos.5,679,377; 5,916,597; 5,912,015; 5,989,463; and 5,128,326; and PCTPublication Nos. WO 99/15154 and WO 99/20253. Examples of polymers usedin sustained release formulations include, but are not limited to,poly(-hydroxy ethyl methacrylate), poly(methyl methacrylate),poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylicacid), polyglycolides (PLG), polyanhydrides, poly(N-vinyl pyrrolidone),poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides(PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In apreferred embodiment, the polymer used in a sustained releaseformulation is inert, free of leachable impurities, stable on storage,sterile, and biodegradable. In yet another embodiment, a controlled orsustained release system can be placed in proximity of the prophylacticor therapeutic target, thus requiring only a fraction of the systemicdose (see, e.g., Goodson, In Medical Applications of Controlled Release,(1984), pages 115-138).

Controlled release systems are discussed in the review by Langer(Science, 249: 1527-1533 (1990)). Any technique known to one of skill inthe art can be used to produce sustained release formulations comprisingone or more therapeutic agents of the invention. See, e.g., U.S. Pat.No. 4,526,938; PCT Publication Nos. WO 91/05548 and WO 96/20698; Ning etal., “Intratumoral Radioimmunotherapy of a Human Colon Cancer XenograftUsing a Sustained-Release Gel,” Radiother. Oncol., 39: 179-189 (1996);Song et al., “Antibody Mediated Lung Targeting of Long-CirculatingEmulsions,” PDA J. Pharm. Sci. Tech., 50: 372-377 (1996); Cleek et al.,“Biodegradable Polymeric Carriers for a bFGF Antibody for CardiovascularApplication,” Proceed. Intl. Symp. Control. Rel. Bioact. Mater., 24:853-854 (1997), and Lam et al., “Microencapsulation of RecombinantHumanized Monoclonal Antibody for Local Delivery,” Proceed. Intl. Symp.Control Rel. Bioact. Mater.: 24: 759-760 (1997), each of which isincorporated herein by reference in their entireties.

In a specific embodiment, where the composition of the invention is anucleic acid encoding a prophylactic or therapeutic agent, the nucleicacid can be administered in vivo to promote expression of its encodedprophylactic or therapeutic agent, by constructing it as part of anappropriate nucleic acid expression vector and administering it so thatit becomes intracellular, e.g., by use of a retroviral vector (see U.S.Pat. No. 4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, DuPont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see, e.g., Joliot et al., Proc. Natl. Acad. Sci. USA,88: 1864-1868 (1991)). Alternatively, a nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression byhomologous recombination.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include, but are not limited to, parenteral (e.g.,intravenous), intradermal, subcutaneous, oral, intranasal (e.g.,inhalation), transdermal (e.g., topical), transmucosal, and rectaladministration. In a specific embodiment, the composition is formulatedin accordance with routine procedures as a pharmaceutical compositionadapted for intravenous, subcutaneous, intramuscular, oral, intranasal,or topical administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocamne to ease pain at the siteof the injection.

If compositions of the invention are to be administered topically, thecompositions can be formulated in the form of an ointment, cream,transdermal patch, lotion, gel, shampoo, spray, aerosol, solution,emulsion, or other form well-known to one of skill in the art. See,e.g., Remington's Pharmaceutical Sciences and Introduction toPharmaceutical Dosage Forms, 19th ed., (Mack Publishing Co., Easton,Pa., 1995). For non-sprayable topical dosage forms, viscous tosemi-solid or solid forms comprising a carrier or one or more excipientscompatible with topical application and having a dynamic viscositypreferably greater than water are typically employed. Suitableformulations include, without limitation, solutions, suspensions,emulsions, creams, ointments, powders, liniments, salves, and the like,which are, if desired, sterilized or mixed with auxiliary agents (e.g.,preservatives, stabilizers, wetting agents, buffers, or salts) forinfluencing various properties, such as, for example, osmotic pressure.Other suitable topical dosage forms include sprayable aerosolpreparations wherein the active ingredient, preferably in combinationwith a solid or liquid inert carrier, is packaged in a mixture with apressurized volatile (e.g., a gaseous propellant, such as FREON®) or ina squeeze bottle. Moisturizers or humectants can also be added topharmaceutical compositions and dosage forms if desired. Examples ofsuch additional ingredients are well known in the art.

If a method of the invention comprises intranasal administration of acomposition, the composition can be formulated in an aerosol form,spray, mist or in the form of drops. In particular, prophylactic ortherapeutic agents for use according to the present invention can beconveniently delivered in the form of an aerosol spray presentation frompressurized packs or a nebulizer, with the use of a suitable propellant(e.g., dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridges(composed of, e.g., gelatin) for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

If a method of the invention comprises oral administration, compositionscan be formulated orally in the form of tablets, capsules, cachets,gelcaps, solutions, suspensions, and the like. Tablets or capsules canbe prepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinised maize starch,polyvinylpyrrolidone, or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose, or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc, or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art. Liquid preparations for oral administration maytake the form of, but not limited to, solutions, syrups or suspensions,or they may be presented as a dry product for constitution with water orother suitable vehicle before use. Such liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, cellulosederivatives, or hydrogenated edible fats); emulsifying agents (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol, or fractionated vegetable oils); andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid). The preparations may also contain buffer salts, flavoring,coloring, and sweetening agents as appropriate. Preparations for oraladministration may be suitably formulated for slow release, controlledrelease, or sustained release of a prophylactic or therapeutic agent(s).

A method of the invention may comprise pulmonary administration, e.g.,by use of an inhaler or nebulizer, of a composition formulated with anaerosolizing agent. See, e.g., U.S. Pat. Nos. 6,019,968; 5,985,320;5,985,309; 5,934,272; 5,874,064; 5,855,913; 5,290,540; and 4,880,078;and PCT Publication Nos. WO 92/19244, WO 97/32572, WO 97/44013, WO98/31346, and WO 99/66903, each of which is incorporated herein byreference their entireties. In a specific embodiment, an antibody of theinvention, combination therapy, and/or composition of the invention isadministered using Alkermes AIR® pulmonary drug delivery technology(Alkermes, Inc., Cambridge, Mass., US).

A method of the invention may comprise administration of a compositionformulated for parenteral administration by injection (e.g., by bolusinjection or continuous infusion). Formulations for injection may bepresented in unit dosage form (e.g., in ampoules or in multi-dosecontainers) with an added preservative. The compositions may take suchforms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle (e.g., sterile pyrogen-free water) before use.

A method of the invention may additionally comprise administration ofcompositions formulated as depot preparations. Such long actingformulations may be administered by implantation (e.g., subcutaneouslyor intramuscularly) or by intramuscular injection. Thus, for example,the compositions may be formulated with suitable polymeric orhydrophobic materials (e.g., as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives (e.g., as asparingly soluble salt).

Methods of the invention encompass administration of compositionsformulated as neutral or salt forms. Pharmaceutically acceptable saltsinclude those formed with anions such as those derived fromhydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., andthose formed with cations such as those derived from sodium, potassium,ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine,2-ethylamino ethanol, histidine, procaine, etc.

Generally, the ingredients of compositions are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the mode of administration is infusion, acomposition can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the mode of administrationis by injection, an ampoule of sterile water for injection or saline canbe provided so that the ingredients may be mixed prior toadministration.

In particular, the invention also provides that one or more of theprophylactic or therapeutic agents or pharmaceutical compositions of theinvention is packaged in a hermetically sealed container such as anampoule or sachette indicating the quantity of the agent. In oneembodiment, one or more of the prophylactic or therapeutic agents, orpharmaceutical compositions of the invention is supplied as a drysterilized lyophilized powder or water free concentrate in ahermetically sealed container and can be reconstituted (e.g., with wateror saline) to the appropriate concentration for administration to asubject. Preferably, one or more of the prophylactic or therapeuticagents or pharmaceutical compositions of the invention is supplied as adry sterile lyophilized powder in a hermetically sealed container at aunit dosage of at least 5 mg, more preferably at least 10 mg, at least15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50 mg,at least 75 mg, or at least 100 mg. The lyophilized prophylactic ortherapeutic agents or pharmaceutical compositions of the inventionshould be stored at between 2° C. and 8° C. in its original containerand the prophylactic or therapeutic agents, or pharmaceuticalcompositions of the invention should be administered within 1 week,preferably within 5 days, within 72 hours, within 48 hours, within 24hours, within 12 hours, within 6 hours, within 5 hours, within 3 hours,or within 1 hour after being reconstituted. In an alternativeembodiment, one or more of the prophylactic or therapeutic agents orpharmaceutical compositions of the invention is supplied in liquid formin a hermetically sealed container indicating the quantity andconcentration of the agent. Preferably, the liquid form of theadministered composition is supplied in a hermetically sealed containerat least 0.25 mg/ml, more preferably at least 0.5 mg/ml, at least 1mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8 mg/ml, at least10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at least 50 mg/ml, atleast 75 mg/ml, or at least 100 mg/ml. The liquid form should be storedat between 2° C. and 8° C. in its original container.

The binding proteins of the invention can be incorporated into apharmaceutical composition suitable for parenteral administration.Preferably, the binding protein will be prepared as an injectablesolution containing 0.1-250 mg/ml antibody. The injectable solution canbe composed of either a liquid or lyophilized dosage form in a flint oramber vial, ampoule or pre-filled syringe. The buffer can be L-histidine(1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0 (optimally pH 6.0). Othersuitable buffers include but are not limited to, sodium succinate,sodium citrate, sodium phosphate or potassium phosphate. Sodium chloridecan be used to modify the toxicity of the solution at a concentration of0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectantscan be included for a lyophilized dosage form, principally 0-10% sucrose(optimally 0.5-1.0%). Other suitable cryoprotectants include trehaloseand lactose. Bulking agents can be included for a lyophilized dosageform, principally 1-10% mannitol (optimally 2-4%). Stabilizers can beused in both liquid and lyophilized dosage forms, principally 1-50 mML-methionine (optimally 5-10 mM). Other suitable bulking agents includeglycine, arginine, can be included as 0-0.05% polysorbate-80 (optimally0.005-0.01%). Additional surfactants include but are not limited topolysorbate 20 and BRIJ surfactants.

The compositions of this invention may be in a variety of forms. Theseinclude, for example, liquid, semi-solid and solid dosage forms, such asliquid solutions (e.g., injectable and infusible solutions), dispersionsor suspensions, tablets, pills, powders, liposomes and suppositories.The preferred form depends on the intended mode of administration andtherapeutic application. Typical preferred compositions are in the formof injectable or infusible solutions, such as compositions similar tothose used for passive immunization of humans with other antibodies. Thepreferred mode of administration is parenteral (e.g., intravenous,subcutaneous, intraperitoneal, intramuscular). In a preferredembodiment, a DLL4 binding protein described herein is administered byintravenous infusion or injection. In another preferred embodiment, aDLL4 binding protein is administered by intramuscular or subcutaneousinjection.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, dispersion, liposome, or other orderedstructure suitable to high drug concentration. Sterile injectablesolutions can be prepared by incorporating the active compound (i.e.,antibody or antibody portion) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the active compound into a sterile vehiclethat contains a basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile,lyophilized powders for the preparation of sterile injectable solutions,the preferred methods of preparation are vacuum drying and spray-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding, in the composition, an agent that delays absorption, forexample, monostearate salts and gelatin.

The DLL4 binding proteins of the present invention can be administeredby a variety of methods known in the art, although for many therapeuticapplications, the preferred route/mode of administration is subcutaneousinjection, intravenous injection, or infusion. As will be appreciated bythe skilled artisan, the route and/or mode of administration will varydepending upon the desired results. In certain embodiments, the activecompound may be prepared with a carrier that will protect the compoundagainst rapid release, such as a controlled release formulation,including implants, transdermal patches, and microencapsulated deliverysystems. Biodegradable, biocompatible polymers can be used, such asethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., (Marcel Dekker, Inc., New York, 1978).

In certain embodiments, a binding protein of the invention may be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The compound (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound of the invention by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, a binding protein of the inventionis coformulated with and/or coadministered with one or more additionaltherapeutic agents that are useful for treating disorders in which DLL4activity is detrimental. For example, an anti-huDLL4 antibody orantibody portion of the invention may be coformulated and/orcoadministered with one or more additional antibodies that bind othertargets (e.g., antibodies that bind other cytokines or that bind cellsurface molecules). Furthermore, one or more binding proteins of theinvention may be used in combination with two or more of the foregoingtherapeutic agents. Such combination therapies may advantageouslyutilize lower dosages of the administered therapeutic agents, thusavoiding possible toxicities or complications associated with thevarious monotherapies.

In certain embodiments, a DLL4 binding protein of the invention islinked to a half-life extending vehicle known in the art. Such vehiclesinclude, but are not limited to, the Fc domain, polyethylene glycol, anddextran. Such vehicles are described, e.g., in U.S. Pat. No. 6,660,843B1 and published PCT Publication No. WO 99/25044, which are herebyincorporated by reference.

In a specific embodiment, nucleic acid sequences comprising nucleotidesequences encoding a binding protein of the invention or anotherprophylactic or therapeutic agent of the invention are administered totreat, prevent, manage, or ameliorate a disorder or one or more symptomsthereof by way of gene therapy. Gene therapy refers to therapy performedby the administration to a subject of an expressed or expressiblenucleic acid. In this embodiment of the invention, the nucleic acidsproduce their encoded binding protein or prophylactic or therapeuticagent of the invention that mediates a prophylactic or therapeuticeffect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. For general reviews of the methodsof gene therapy, see Goldspiel et al., Clin. Pharmacy, 12: 488-505(1993); Wu and Wu, Biotherapy, 3: 87-95 (1991); Tolstoshev, Ann. Rev.Pharmacol. Toxicol., 32: 573-596 (1993); Mulligan, Science, 260: 926-932(1993); and Morgan and Anderson, Ann. Rev. Biochem., 62: 191-217 (1993);Robinson, C., Trends Biotechnol., 11(5):155 (1993). Methods commonlyknown in the art of recombinant DNA technology which can be used aredescribed in Ausubel et al. (eds.), Current Protocols in MolecularBiology (John Wiley & Sons, New York, 1993); and Kriegler, Gene Transferand Expression, A Laboratory Manual, (Stockton Press, New York, 1990).Detailed descriptions of various methods of gene therapy are disclosedin US Patent Application Publication No. 20050042664 A1, which isincorporated herein by reference.

In another aspect, this invention provides a method of treating (e.g.curing, suppressing, ameliorating, delaying, or preventing the onset of,or preventing recurrence or relapse of) or preventing a DLL4-associatedtumor in a subject. The method includes administering to a subject aDLL4 binding protein, e.g., an anti-DLL4 antibody or fragment thereof asdescribed herein, in an amount sufficient to treat or prevent theDLL-associated tumor or cancer. The DLL4 antagonist, i.e., the anti-DLL4antibody or fragment thereof, may be administered to a subject alone orin combination with other therapeutic modalities as described herein.

DLL4 plays a critical role in the pathology associated with a variety ofdiseases involving immune and inflammatory elements, in particularcancer and tumor angiogenesis. Examples of DLL4-associated disordersinclude, but are not limited to, those disorders that adversely affectthe following biological processes: neuronal function and development;stabilization of arterial endothelial fate and angiogenesis; regulationof crucial cell communication events between endocardium and myocardiumduring both the formation of the valve primordial and ventriculardevelopment and differentiation; cardiac valve homeostasis, as well asimplications in other human disorders involving the cardiovascularsystem; timely cell lineage specification of both endocrine and exocrinepancreas; influencing of binary fate decisions of cells that must choosebetween the secretory and absorptive lineages in the gut; expansion ofthe hematopoietic stem cell compartment during bone development andparticipation in commitment to the osteoblastic lineage such asosteoporosis; regulation of cell-fate decision in mammary glands atseveral distinct development stages; and certain non-nuclear mechanisms,such as control of the actin cytoskeleton through the tyrosine kinaseAbl. More specifically, DLL4-associated disorders include, but are notlimited to, cancers, T-ALL (T-cell acute lymphoblastic leukemia),CADASIL (cerebral autosomal dominant arteriopathy with subcorticalinfarcts and leukoencephalopathy), MS (multiple sclerosis), tetralogy ofFallot (TOF), and Alagille syndrome (AS), macular degeneration andage-related macular degeneration diseases, and other angiogenesisindependent and dependent diseases characterized by aberrant DLL4expression or activity.

Preferably, DLL4 binding proteins, such as antibodies andantigen-binding portions thereof as described herein, are used to treatcancers and tumors.

Binding proteins according to the invention can be used alone or incombination, i.e., more than one DLL4-binding protein described herein,to treat a cancer, a tumor, or other disorder in which binding to,inhibition of, and/or neutralization of DLL4 is considered desirable orotherwise beneficial to the health of an individual.

It should be understood that DLL4 binding proteins of the invention canalso be used alone or in combination with an additional agent, e.g., atherapeutic agent, said additional agent being selected by the skilledpractitioner for its intended purpose. For example, the additional agentcan be a therapeutic agent that is recognized in the art as being usefulto treat a cancer, tumor, or other disease or condition in which bindingto or inhibition of DLL4 is considered to be desirable or advantageousfor treating the cancer, tumor, or other disease or condition. Theadditional agent also can be an agent that imparts a beneficialattribute to the therapeutic composition, e.g., an agent which affectsthe viscosity of the composition.

It should further be understood that the combinations which are to beincluded within this invention are those combinations useful for theirintended purpose. The agents set forth below are illustrative forpurposes and not intended to be limited. The combinations, which arepart of this invention, can be the antibodies of the present inventionand at least one additional agent selected from the lists below. Thecombination can also include more than one additional agent, e.g., twoor three additional agents, if the combination is such that the formedcomposition can perform its intended function.

Preferred combinations of therapeutic agents may interfere at differentpoints in the pro-tumorigenic or pro-angiogenic signaling pathways.Preferred examples of therapeutic agents useful in the methods andcompositions of the invention include antineoplastic agents,radiotherapy, and chemotherapy such as DNA alkylating agents, cisplatin,carboplatin, anti-tubulin agents, paclitaxel, docetaxel, taxol,doxorubicin, gemcitabine, gemzar, anthracyclines, adriamycin,topoisomerase I inhibitors, topoisomerase II inhibitors, 5-fluorouracil(5-FU), leucovorin, irinotecan, receptor tyrosine kinase inhibitors(e.g., erlotinib, gefitinib), COX-2 inhibitors (e.g., celecoxib), andkinase inhibitors.

The DLL4 binding proteins of the invention may also be administered incombination with agents, such as methotrexate, 6-MP, azathioprinesulphasalazine, mesalazine, olsalazine chloroquinine/hydroxychloroquine,pencillamine, aurothiomalate (intramuscular and oral), azathioprine,colchicine, corticosteroids (oral, inhaled and local injection), beta-2adrenoreceptor agonists (salbutamol, terbutaline, salmeteral), xanthines(theophylline, aminophylline), cromoglycate, nedocromil, ketotifen,ipratropium and oxitropium, cyclosporin, FK506, rapamycin, mycophenolatemofetil, leflunomide, NSAIDs, for example, ibuprofen, corticosteroidssuch as prednisolone, phosphodiesterase inhibitors, adensosine agonists,antithrombotic agents, complement inhibitors, adrenergic agents, agentswhich interfere with signaling by proinflammatory cytokines such as TNFαor IL-1 (e.g. IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1βconverting enzyme inhibitors, TNFα converting enzyme (TACE) inhibitors,T-cell signaling inhibitors such as kinase inhibitors, metalloproteinaseinhibitors, sulfasalazine, azathioprine, 6-mercaptopurines, angiotensinconverting enzyme inhibitors, soluble cytokine receptors and derivativesthereof (e.g., soluble p55 or p75 TNF receptors and the derivativesp75TNFRIgG (Enbrel™) and p55TNFRIgG (Lenercept), sIL-1RI, sIL-1RII,sIL-6R), antiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 andTGFβ), celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib,etanercept, infliximab, naproxen, valdecoxib, sulfasalazine,methylprednisolone, meloxicam, methylprednisolone acetate, gold sodiumthiomalate, aspirin, triamcinolone acetonide, propoxyphenenapsylate/apap, folate, nabumetone, diclofenac, piroxicam, etodolac,diclofenac sodium, oxaprozin, oxycodone hcl, hydrocodonebitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra,human recombinant, tramadol hcl, salsalate, sulindac,cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium,prednisolone, morphine sulfate, lidocaine hydrochloride, indomethacin,glucosamine sulf/chondroitin, amitriptyline HCl, sulfadiazine, oxycodoneHCl/acetaminophen, olopatadine hcl, misoprostol, naproxen sodium,omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18BP, anti-IL-18, anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740,Roflumilast, IC-485, CDC-801, and Mesopram.

Non-limiting examples of therapeutic agents for cancers with which aDLL4 binding protein of the invention can be co-administered or used incombination include the following: budenoside; epidermal growth factor;sulfasalazine; aminosalicylates; 6-mercaptopurine; azathioprine;metronidazole; lipoxygenase inhibitors; mesalamine; olsalazine;balsalazide; antioxidants; thromboxane inhibitors; IL-1 receptorantagonists; anti-IL-1β monoclonal antibodies; anti-IL-6 monoclonalantibodies; growth factors; elastase inhibitors; pyridinyl-imidazolecompounds; and antibodies to or antagonists of other human cytokines orgrowth factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8,IL-15, IL-16, IL-17, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Antibodiesof the invention, or antigen binding portions thereof, can be combinedwith antibodies to cell surface molecules such as CD2, CD3, CD4, CD8,CD25, CD28, CD30, CD40, CD45, CD69, CD90, or their ligands.

Other examples of therapeutic agents with which a DLL4 binding proteinof the invention can be combined include the following: TNF antagonists,for example, anti-TNF antibodies, D2E7 (PCT Publication No. WO 97/29131;HUMIRA®), CA2 (REMICADE®), CDP 571, TNFR-Ig constructs (p75TNFRIgG(ENBREL®) and p55TNFRIgG (LENERCEPT)), and PDE4 inhibitors. Bindingproteins of the invention can be combined with mesalamine, prednisone,azathioprine, mercaptopurine, infliximab, methylprednisolone sodiumsuccinate, diphenoxylate/atrop sulfate, loperamide hydrochloride,methotrexate, omeprazole, folate, ciprofloxacin/dextrose-water,hydrocodone bitartrate/apap, tetracycline hydrochloride, fluocinonide,metronidazole, thimerosal/boric acid, cholestyramine/sucrose,ciprofloxacin hydrochloride, hyoscyamine sulfate, meperidinehydrochloride, midazolam hydrochloride, oxycodone hcl/acetaminophen,promethazine hydrochloride, sodium phosphate,sulfamethoxazole/trimethoprim, celecoxib, polycarbophil, propoxyphenenapsylate, hydrocortisone, multivitamins, balsalazide disodium, codeinephosphate/apap, colesevelam hcl, cyanocobalamin, folic acid,levofloxacin, methylprednisolone, natalizumab, and interferon-gamma.

Non-limiting examples of therapeutic agents with which a binding proteinof the invention can be combined include the following: aspirin,nitroglycerin, isosorbide mononitrate, metoprolol succinate, atenolol,metoprolol tartrate, amlodipine besylate, diltiazem hydrochloride,isosorbide dinitrate, clopidogrel bisulfate, nifedipine, atorvastatincalcium, potassium chloride, furosemide, simvastatin, verapamil hcl,digoxin, propranolol hydrochloride, carvedilol, lisinopril,spironolactone, hydrochlorothiazide, enalapril maleate, nadolol,ramipril, enoxaparin sodium, heparin sodium, valsartan, sotalolhydrochloride, fenofibrate, ezetimibe, bumetanide, losartan potassium,lisinopril/hydrochlorothiazide, felodipine, captopril, and bisoprololfumarate.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a binding protein of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the binding protein may bedetermined by a person skilled in the art and may vary accordingtofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the binding protein to elicit a desiredresponse in the individual. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the binding protein areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous toformulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a DLL4 binding protein of theinvention is 0.1-20 mg/kg, more preferably 1-10 mg/kg. It is to be notedthat dosage values may vary with the type and severity of the conditionto be alleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

III. Use in Immunotechniques.

Any of a variety of immunodetection assay formats may be adapted toemploy a DLL4 binding protein of the invention for use in detecting DLL4present in a mixture, solution, or biological sample. Suchimmunodetection assay formats include but are not limited toradioimmunoassay (RIA), immunoprecipitation, enzyme-linked immunosorbentassay (ELISA), immunoblot (e.g., Western blot), immunostrips (e.g.,immunodipsticks) that comprise a DLL4 binding protein of the inventionadsorbed or immobilized to substrate, fluorescence activated cellsorting (FACS), and the like. A DLL4 binding protein described hereincan be adsorbed or immobilized to a substrate, e.g., a resin particle orother material, for use in an affinity column or any other affinityformat available in the art to purify DLL4 from a sample. Detection ofDLL4 using a DLL4 binding protein of the invention can be conducted invitro on a mixture, solution, or in biological sample. A biologicalsample that can be contacted with a DLL4 binding protein of theinvention to detect or measure DLL4 in the sample includes, but is notlimited to, urine, saliva, oral swab (buccal, lingual, or throat swab),dermal swab, dermal scrape, rectal swab, vaginal swab, whole bloodsample, plasma sample, serum sample, tissue biopsy, and any other sampleobtained from an individual by a procedure known in the art. In anotherembodiment, a DLL4 binding protein may be employed to detect DLL4 invivo such as various tomography and scanning methods, including but notlimited to X-ray computer assisted tomography (CT), magnetic resonanceimaging (MRI), and positron emission tomography (PET).

It will be readily apparent to those skilled in the art that othersuitable modifications and adaptations of the methods of the inventiondescribed herein are obvious and may be made using suitable equivalentswithout departing from the scope of the invention or the embodimentsdisclosed herein. Having now described the present invention in detail,the same will be more clearly understood by reference to the followingexamples, which are included for purposes of illustration only and arenot intended to be limiting of the invention.

EXAMPLES Example 1: In Vitro Assays Used to Determine the FunctionalActivity of DLL4 Antibodies Example 1.1: Affinity Determination UsingBIACORE® Surface Plasmon Resonance Technology

The BIACORE® surface plasmon resonance assay (Biacore, Inc., Piscataway,N.J., US) determines the affinity of antibodies with kineticmeasurements of on-rate and off-rate constants. Binding of DLL4antibodies to a purified recombinant DLL4 extracellular domain (ECD) wasdetermined by surface plasmon resonance-based measurements with aBiacore® instrument (either a Biacore 2000, Biacore 3000, or BiacoreT100; GE Healthcare, Piscataway, N.J., US) using running buffer HBS-EPB(10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, 0.1 mg/ml BSA and 0.005%surfactant P20) at 25° C. For example, approximately 9000 RU of goatanti-human Fc specific polyclonal antibody (Thermo Fisher ScientificInc., Rockford, Ill., US) diluted in 10 mM sodium acetate (pH 4.5) isdirectly immobilized across a CMS research grade biosensor chip using astandard amine coupling kit according to manufacturer's instructions andprocedures at 25 μg/ml. Unreacted moieties on the biosensor surface wereblocked with ethanolamine. For kinetic analysis, rate equations derivedfrom the 1:1 Langmuir binding model were fitted simultaneously tomultiple antigen injections (using global fit analysis) with the use ofScrubber 2 (BioLogic Software), Biacore Biaevaluation 4.0.1 software orBiacore T100 Evaluation software. Purified antibodies were diluted inrunning buffer for capture across goat anti-human Fc reaction surfaces.Antibodies to be captured as a ligand (1 μg/ml) were injected overreaction matrices at a flow rate of 10 μl/min. During the assay, allmeasurements were referenced against the capture surface alone (i.e.,with no captured anti-DLL4 antibody). The association and dissociationrate constants, K_(on) (M⁻¹s⁻¹) and K_(off) (s⁻¹) were determined undera continuous flow rate of 80 μl/min. Rate constants were derived bymaking kinetic binding measurements at different antigen concentrationsranging from 1.23-900 nM, as a 3-fold dilution series, and includedbuffer-only injections (to be used for double referencing). Theequilibrium dissociation constant K_(D) (M) of the reaction betweenantibodies and the target antigen was then calculated from the kineticrate constants by the following formula: K_(D)=K_(off)/K_(on). Bindingwas recorded as a function of time and kinetic rate constants werecalculated. In this assay, on-rates as fast as 10⁶ M^(−l)s⁻¹ andoff-rates as slow as 10⁻⁶ s⁻¹ could be measured.

Example 1.2: Binding of DLL4 Antibodies to Soluble DLL4 ExtracellularDomain as Determined by ELISA Method 1 (Capture ELISA).

96-well Nunc-Immuno plates (#439454) were coated with 5 μg/ml antibodyagainst human IgG (Fcg fragment specific, Jackson ImmunoResearch,#109-005-098, 100 μl/well) in D-PBS (Gibco #14190) and incubatedovernight at 4° C. ELISA plates were washed 3 times with wash buffer(PBS, 0.05% Tween-20) and then blocked with 200 ml/well blocking buffer(D-PBS, 1% BSA, 1 mM CaCl₂, 0.05% Tween-20) for 1 hour at 25° C. Plateswere washed 3 times and incubated with 100 μl/well DLL4 antibodies(0.0001-100 nM, 10-fold serial dilution in blocking buffer) for 1 hourat 25° C., and then washed again 3 times. Plates containing capturedDLL4 antibody were incubated with biotin-labeled human DLL4extracellular domain (10 nM in blocking buffer, 100 μl/well) for 1 hourat 25° C., washed 3 times, and incubated with streptavidin conjugatedwith HRP (KPL #474-3000, 1:10,000 dilution in blocking buffer, 100μl/well) for 1 hour at 25° C. After the final wash, plates wereincubated with 100 μl/well ELISA substrate (1-Step Ultra TMB-ELISA,Pierce #340280). The reaction was stopped after 2 minutes at 25° C. with100 μl/well 2 N H2504 and the absorbance was read at 450 nm. Data wereanalyzed using Graphpad Prism software and EC₅₀ values were reported.

Method 2 (Copper Coated Plate).

96-well copper-coated plates (Thermo Scientific #15143) were washed 3times with wash buffer (PBS, 0.05% Tween-20) before use and thenincubated with 100 μl/well of 6×His-tagged recombinant DLL4extracellular domain (ECD) (“6×His” disclosed as SEQ ID NO: 206) at 1μg/ml in PBS, 1 hour at 25° C. with shaking. Plates were then washed 3times. 100 μl/well of recombinant rat/human chimeric or recombinanthuman anti-DLL4 antibodies were then added to the plate (0.00164-27 nM,4-fold serial dilution in ELISA buffer=PBST, 10% Superblock (Pierce#37515)) for 1 hour at 25° C. with shaking and then washed again 3times. Plates were incubated with goat anti-human HRP (Pierce #31412)(1:40,000 dilution in ELISA buffer, 100 μl/well) for 1 hour at 25° C.with shaking, then washed 3 times. After the final wash, plates wereincubated with 100 μl/well ELISA substrate (Sigma #T8665). The reactionwas stopped after 8 minutes at 25° C. with 100 μl/well 1N HCl and theabsorbance was read at 450 nm. Data were analyzed using Graphpad Prismsoftware, and EC50 values were reported.

Example 1.3: Binding of DLL4 Monoclonal Antibodies to the Surface ofHuman Tumor Cell Lines as Assessed by Flow Cytometry (FACS)

Stable cell lines overexpressing cell-surface DLL4 were harvested fromtissue culture flasks, washed four times and resuspended in phosphatebuffered saline (PBS) containing 1% bovine serum albumin and 1 mM CaCl₂(FACS buffer). 1.5×10⁵ cells were incubated with antibodies at variousconcentrations in FACS buffer for 60 minutes on ice. Cells were washedtwice and 504 of R-phycoerythrin-conjugated anti-rat IgG, F(ab′)₂fragment (1:200 dilution in FACS buffer) (Jackson ImmunoResearch, WestGrove, Pa., Cat. #112-116-072) were added. Following an incubation onice (4° C., 60 minutes), cells were washed three times and resuspendedin FACS buffer. Fluorescence was measured using a Becton DickinsonFACSCalibur-HTS (Becton Dickinson, San Jose, Calif., US). Data wereanalyzed using Graphpad Prism software and EC₅₀ values were reported asthe concentration of antibody to achieve 50% of maximal DLL4 antibodiesbinding to DLL4 expressing cells.

Example 1.4: Inhibition of Notch-1 Interaction with Soluble DLL4Extracellular Domain by DLL4 Antibodies (Competition ELISA)

96-well Nunc-Immuno plates (#439454 for huDLL4 ELISA) and 96-well Costarplates (#9018 for muDLL4 ELISA) were coated with 16 nM human Notch-1(R&D Systems #3647-TK, 100 μl/well in D-PBS) and incubated overnight at4° C. Plates were then washed 3 times with wash buffer (PBS, 0.05%Tween-20) and blocked with 200 μl/well blocking buffer (D-PBS, 1% BSA, 1mM CaCl₂, 0.05% Tween-20) for 1 hour at 25° C. While blocking, biotinlabeled DLL4 extracellular domain (14 nM) was mixed with antibody (30pM-66 nM, 3-fold serial dilution in blocking buffer) for 1 hour at 25°C. with shaking. Assay plates were washed after blocking, and incubatedwith DLL4/antibody mixtures (100 μl/well, 1 hour at 25° C. withshaking). Plates were washed again and 100 μl/well streptavidinconjugated with HRP (Fitzgerald #65R-S104PHRPx, diluted 1:5,000 inblocking buffer) was added for 1 hour at 25° C. with shaking. After afinal wash, plates were developed using 100 μl/well substrate (TMB Sigma#T8665), and the reaction was stopped using 100 μl/well 1N HCl, and theabsorbance was read at 450 nm. Data were analyzed using Graphpad Prismsoftware and IC₅₀ values were reported as the concentration of antibodyto achieve 50% reduction of DLL4 bound to Notch1.

Example 1.5: Blocking of Soluble Notch Binding to DLL4-Overexpressing293G Cells by Anti-DLL4 Monoclonal Antibodies as Assessed by FlowCytometry (Competition FACS)

Notch blocking assay: Briefly, stable cell lines overexpressingcell-surface DLL4 were harvested from tissue culture flasks andre-suspended in phosphate buffered saline (PBS) containing 1% bovineserum albumin and 1 mM CaCl₂ (FACS buffer). HEK293G-DLL4 cells weredispensed into 96-well plate (v-bottom) at 1.5×10⁵ cells/well in FACSbuffer. After spinning down cells and discarding the supernatant, 50 μLof purified IgG with appropriate dilution were added to each well, andincubated on ice at 4° C. for 60 minutes, followed by addition of 50μL/well of Notch1-biotin at 0.2 μg/mL for human DLL4-293G or 2.0 μg/mLfor mouse DLL4-293G (1.0 or 0.1 μg/mL final) for an additional 1 hourincubation ice at 4° C. After washing the cells two times with FACSbuffer, 50 μL of R-phycoerythrin-conjugated streptavidin (1:150 dilutionin FACS buffer) (Jackson ImmunoResearch, West Grove, Pa., Cat.#016-110-084) were added. Following an incubation on ice (4° C., 60minutes), cells were washed three times and resuspended in FACS buffer.Fluorescence was measured using a Becton Dickinson FACSCalibur-HTS(Becton Dickinson, San Jose, Calif.). Data were analyzed using GraphpadPrism software, and IC₅₀ values were reported as the concentration ofantibody to achieve 50% reduction of Notch1 bound to DLL4 expressingcells.

Example 1.6: Inhibition of DLL4-Dependent Notch Activation in EA.hy926Cells by DLL4 Antibodies Using Notch Reporter Assay

96-well black clear-bottom tissue culture plates were seeded overnightwith 7,000 cells/well engineered EA.hy926 cells expressing luciferasedriven by a Notch-responsive promoter. Antibodies serially diluted from200 nM were mixed for 15 minutes with equal volume of 5,000 HEK293Gcells/well expressing full-length DLL4. The 293G/DLL4 cells wereco-cultured with EA.hy926 Notch reporter cells for 24 hours in thepresence of testing antibodies. Luciferase activity was analyzed byPromega's substrate (Promega #E2940). Data was analyzed using GraphpadPrism software, and IC₅₀ values were reported as the concentration ofantibody to achieve 50% reduction of DLL4-induced Notch activation.

Example 1.7: Analytical Methods and Techniques for PhysicochemicalProperty Characterizations PEG Precipitation Method.

The use of PEG for inducing phase separation of a solid proteinaccording to principles of volume exclusion represents a feasibleapproach to assess the solubility of a protein. PEG has severaladvantages over other precipitants, including minimal denaturation ofproteins at ambient temperatures (does not affect tertiary structure ofproteins) and within the range of 4° C. to 30° C. temperature control isnot required, i.e., precipitation studies can be performed at ambienttemperature at the laboratory bench.

Generally, the precipitation of proteins by PEGs is explained on thebasis of volume exclusion effects. According to this theory, proteinsare sterically excluded from the regions of solvent that are occupied byPEG linear chains. As a result, proteins are concentrated and eventuallyprecipitated when their solubility is exceeded. In thermodynamic terms,the steric exclusion leads to an increase in the chemical potential ofthe protein until it exceeds that of the pure solid state, resulting inprotein precipitation. This happens mainly because of a largeunfavorable free energy of interaction between PEG and proteins,reducing the preferential hydration of protein due to steric exclusioneffects. In aqueous solutions, preferential hydration helps to maintainthe native structure of proteins. Generally, volume exclusion has beenshown to become more effective with increasing molecular weight of thePEG, i.e., less PEG is needed to precipitate proteins with increasingPEG molecular weight.

A PEG molecular weight of 3000 was chosen for estimating the solubilityof the antibodies covered by this patent. A 50% PEG solution was made bydissolving PEG in deionized water in the ratio of one gram of PEG to 1mL of water. The PEG solution was then added to a solution of antibodywhich was initially at a concentration of less than or equal to 0.5mg/ml and a volume of 0.5 mL. The PEG solution was continually added andmixed until the first instance of cloudiness persists. The percentage ofPEG 3000 needed to cause this precipitation is calculated as(50)×(volume of PEG 3000 solution added)/(initial volume of antibodysolution before PEG addition).

The percentage of PEG 3000 needed for precipitation was compared to thepercentage needed for precipitation of protein with known watersolubility. For example, the water solubility of adalimumab exceeds 200mg/mL. Consequently, if the percentage of PEG 3000 required toprecipitate a protein of interest is similar to the percentage needed toprecipitate adalimumab, then the predicted solubility of that proteinwill be similar to the solubility adalimumab.

Real Solubility Method.

Real solubility is determined by using Amicon centrifugal filters toconcentrate a protein in solution until the protein is observed toprecipitate out of solution or until the minimum volume to which theprotein can be concentrated within the filter unit is reached. For thelatter, 15 mL Amicon centrifugal filters have a minimum volume of ˜50 μlwhile 4 mL Amicon centrifugal filters have a minimum volume of ˜15 μl.

First, a protein was dialyzed into a specific formulation(s). For thesestudies, the antibody amount was 10 mg or much less. Then the proteinsolution was inserted into the Amicon centrifugal filter retentatechamber. The chamber was lined with a nitrocellulose membrane with poresthat permitted molecules of less than 10 to 30 kilodaltons to pass whensubjected to centrifugal force. Antibodies, which were typically above140 kilodaltons, were retained while water, buffer molecules, smallexcipients, and salts passed through. The centrifugal filter was thencentrifuged according to manufacturer specifications until the proteinwas observed to precipitate out of solution or until the minimum volumeto which the protein can be concentrated within the filter unit wasreached.

After centrifugation, the protein solution was removed from theretentate chamber, and the concentration was measured by ultravioletabsorbance. The solution was then kept at 25° C. and 5° C. for 1 to 2days and was monitored for signs of precipitation. Near UV-CD Technique.

Near UV-CD spectroscopy provides important information about thetertiary structure of proteins and was one of the most used techniquesin this regards. CD refers to the differential absorption of the leftand right circularly polarized components of plane polarized radiation.For proteins, the chromophores in the near UVCD region (250-320 nm) arethe aromatic amino acids (i.e., tryptophan, tyrosine, and phenylalanine)and the disulfide bonds, and the CD effect occurs when the chromophoresare present in an asymmetric (buried) environment. Signals in the regionfrom 250-270 nm are attributable to phenylalanine residues, signals from270-290 nm are attributable to tyrosine, and those from 280-300 nm areattributable to tryptophan. Disulfide bonds give rise to broad weaksignals throughout the near-UV spectrum. The near-UV CD spectrum can besensitive to small changes in tertiary structure such as those due toprotein-protein interactions and/or changes in formulation conditions.

There are a number of other factors that can influence the CD spectra ofaromatic amino acids. Among these are (1) the rigidity of the protein,(2) the nature of hydrogen bonding, and (3) interactions between variousaromatic amino acids. Additionally, proteins with large number of suchamino acids can have smaller CD bands due to the cancellation of thepositive and negative bands.

Briefly, a protein dialyzed into the desired formulation(s) at 1 mg/mland was scanned from 250-320 nm or 240-320 nm with a Jasco 800 CDspectrometer. The corresponding formulation without protein was alsoscanned, and the readings subtracted from that of the scan of theprotein solution. A near UV-CD spectra was a plot of molar ellipticitiesversus wavelength from 250 or 240 to 320 nm.

For antibodies in general, a near UV-CD spectrum with a semi-sigmoidalprofile indicates good tertiary structure folding while a flatter andless featured profile indicates a greater tendency to unfold. Compactfolding is associated with good stability while poor folding exposes thehydrophobic interior which may lead to hydrophobic interactions amongprotein molecules resulting in the formation of undesired aggregates.

DSC Technique.

The thermal stability of the antibodies was assessed using adifferential scanning calorimetry (DSC) instrument. The DSC instrumentused was an automated VP-DSC equipment with Capillary Cell (Microcal, GEHealthcare Ltd./Microcal, Buckinghamshire, UK). Unfolding of moleculeswas studied applying a 1° C./minute scan rate over a 25° C.-95° C.temperature range for samples at 1 mg/mL. Additional measurementparameters applied were a fitting period of 16 seconds, a pre-scan waittime of 10 minutes, and measurements were performed in none-feedbackmode. Per individual measurement, 420 μL of sample/blank were filledinto the DSC measurement sample holder, with a plate fill scheme asprovided below. The thermograms obtained were fitted to a non two statemodel to obtain the midpoint temperatures and enthalpies of thedifferent transitions.

An additional requirement for successful biologics development candidateis that the protein remains in its native state and conformation. Aprotein in aqueous solution is in equilibrium between the native(folded) conformation and its denatured (unfolded) conformation. Thestability of the native state is based on the magnitude of the Gibbsfree energy (DG) of the system and the thermodynamic relationshipbetween enthalpy (DH) and entropy (DS) changes. A positive DG indicatesthe native state is more stable than the denatured state—the morepositive the DG, the greater the stability. For a protein to unfold,stabilizing forces need to be broken. Conformational entropy overcomesstabilizing forces allowing the protein to unfold at temperatures whereentropy becomes dominant. DSC measures DH of protein unfolding due toheat denaturation. As a general rule, it can be stated that the higherthe transition midpoint (the Tm), the more stable the protein at lowertemperatures. During the same experiment, DSC also measures the changein heat capacity (DCp) for protein denaturation. Heat capacity changesassociated with protein unfolding are primarily due to changes inhydration of side chains that were buries in the native state, butbecome solvent exposed in the denatured state. DSC has been shown to bea valuable predictor of liquid formulation stability for proteins andother biological macromolecules (Remmele, R. L. Jr., Gombotz, W. R.,BioPharm 13, 36-46, 2000, and; Remmele, R. L. Jr., Nightlinger, N. S.,Srinivasen, S., Gombotz, W. R., Pharm. Res. 15, 200-208, 1998).

SEC Technique.

Size exclusion chromatography (SEC) was used to separate proteins basedon size. Proteins are carried in an aqueous mobile phase and through aporous stationary phase resin packed in a column. The retention time inthe column is a function of the hydrodynamic size of the protein and thesize of the pores in the packed resin bed. Smaller molecules canpenetrate into smaller pores in the resin and are retained longer thanlarger molecules. Upon elution from the column, the proteins aredetected by UV absorbance. The SEC method used a TSK gel guard (TOSOHBiosciences, Montgomeryville, Pa., cat. no. 08543) and a TSK gelG3000SWxL (TOSOH Biosciences, Montgomeryville, Pa., cat. no. 08541). Themobile phase was 100 mM Na₂HPO₄, 200 mM Na₂SO₄, pH 6.8. The flow ratewas 0.25 mL/minute. Injection volume was 20 μL of 1 mg/mL sample. Thecolumn temperature was room temperature. The autosampler temperature was2-8° C. The total run time was 55 minutes. The detection was based on UVabsorbance at 214 nm wavelength, with band width set at 8 nm, usingreference wavelength at 360 nm with band width 100 nm.

Freeze-Thaw Method.

Antibody solutions at 1 mg/ml in the desired formulation(s) were frozenat −80° C. for at least 4 hours and then thawed at 30° C. in a waterbath. The solutions were then refrozen at −80° C. This was repeated for5 cycles. After certain freeze-thaw cycles, e.g., second and fourth, aportion of the solution was withdrawn for analysis by SEC beforerefreezing. Freeze-thaw stability testing was done at low proteinconcentration in order obtain a “worse-case scenario” due to greaterexposure of protein molecules to the denaturing ice-water interfaces. Athigher concentrations, proportionally less protein encounters theice-water interface, instead interacting with other protein molecules.

Accelerated Stability Method.

Antibody solutions at 1 mg/ml in the desired formulation(s) were passedthrough 0.22 μm PVDF filters under sterile conditions and incubated at40° C. and/or 50° C. for at least 21 days. At 7 days and 21 days,aliquots were withdrawn under sterile conditions and subjected toanalysis by SEC. Solutions were then returned to incubation.

Example 2: Generation of Rat Anti-DLL4 Monoclonal Antibodies by RatHybridoma Technology

Rats were immunized according to the methods known in the art (forexample, E Harlow, D. Lane, Antibody: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1998). A human cellline expressing full-length human DLL4 as well as recombinant mouseDLL4-ECD (DLL4 extracellular domain) proteins was used as immunogen.Mouse cell lines expressing either human DLL4 or mouse DLL4 were usedfor determining anti-sera titer and for screening hybridomas secretingantigen-specific antibodies. Immunizing dosages contained 1×10⁶cells/rat/injection for both primary and boost immunizations. Toincrease immune response to mouse DLL4, the rats were further boostedwith recombinant mouse DLL4-ECD in emulsion form with an incompleteFreud's adjuvant (Sigma, St. Louis, Mo., US). Briefly, adjuvant-antigenmixture was prepared by first gently mixing the adjuvant in a vial usinga vortex. The desired amount of adjuvant was removed from the vial andput into an autoclaved 1.5 mL microcentrifuge tube. The antigen wasprepared in PBS or saline with concentration ranging from 0.5-1.0 mg/ml.The calculated amount of antigen was then added to the microcentrifugetube with the adjuvant, and the solution was mixed by gently vortexingfor 2 minutes to generate water-in-oil emulsion. The adjuvant-antigensolution was then drawn into the proper syringe for animal injection. Atotal of 50 μg of antigen was injected in a volume of 50-100 μl. Eachanimal was immunized, and then boosted for 2 to 3 times depending on thetiter. Animals with good titers were given a final subcutaneous boostwith cell line expressing human DLL4 before fusion.

Hybridoma Fusion and Screening.

Cells of murine myeloma cell line (SP2/0-Ag14, ATCC CRL-1581) werecultured to reach the log phase stage right before fusion. Immunized ratspleen cells were prepared sterilely and fused with myeloma cellsaccording to the methods known in the art (for example, E Harlow, D.Lane, Antibody: A Laboratory Manual, (Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1998); Kohler G, and Milstein C.,“Continuous cultures of fused cell secreting antibody of predefinedspecificity,” Nature, 256: 495-497 (1975)). Fused “hybrid cells” weresubsequently dispensed into 96-well plates in DMEM/20% FCS/HAT media.Surviving hybridoma colonies were observed under the microscope seven toten days post-fusion. After two weeks, the supernatant from each wellwas subjected to cell-based binding screening using mouse cell linesexpressing either recombinant human DLL4 or mouse DLL4. Briefly, themixture (1:1 ratio) of mouse cell lines expressing either human or mouseDLL4 was dispensed into a 96-well (round bottom) plate at 1×10⁶cells/well and incubated with hybridoma supernatant (50 μl) at 4° C. for1 hour. Cells were then washed 3 times with FACS buffer (PBS+2% BSA),HRP-goat anti-rat Ig-PE (phycoerythrin) was used for detection in FACSmachine. Hybridomas were screened using ELISA format according thefollowing procedure. ELISA plates were coated with 50 μl of either humanDLL4 or mouse DLL4 (2.0 μg/ml in PBS) overnight at 4° C. Plates werewashed 3 times with 250 μl PBS/0.5% Tween₂₀ and blocked with 200 μlblocking buffer (2% BSA in PBS with 0.5% Tween₂₀). Diluted sera orhybridoma supernatant (100 μl) was added to each well, and incubated atroom temperature for 1 hour. Plates were then washed 3 times withPBS/0.5% Tween₂₀, HRP-goat anti-rat-IgG was used for detection, andbinding ODs were observed at 450 nm. Positive hybridoma secretingantibody that binds to either human DLL4 or mouse DLL4 or both were thenselected and transferred to 24-well plates and subcloned by limitingdilution to ensure the clonality of the cell line. The isotype of eachmonoclonal antibody was determined using the Zymed's Mouse MonoAb-IDKit. Hybridoma clones producing antibodies that showed high specificbinding activity were subcloned and purified (Table 5), and affinity(Biacore) and potency (Notch blocking FACS and reporter assay) of theantibodies were characterized as follows.

TABLE 5 A List of Anti-DLL4 Antibodies Generated Using Rat HybridomaTechnology FACS binding Human Mouse Hybridoma Isotype DLL4 DLL4MC10-37D10.1C2 IgG2a/k + + MC10-40B10.3C3 IgG2a/k + + MC10-32C7.5A4IgG2a/k + + MC10-38H12.2G8 IgG2a/k + − MC13-14A11.3A4 IgG2a/K + +MC13-14G1.1B4 IgG2a/K + − MC13-1A11.2E1 IgG1/K + + MC13-13E4.4A3IgG1/K + + MC13-15D6.1G7 IgG2a/K + + “+” indicates antibody bound tocells; “−” indicates antibody did not bind to cells

Example 3. In Vitro Characterization of Anti-DLL4 Rat MonoclonalAntibodies

The antigen binding affinities of these rat monoclonal antibodies (mAbs)were determined by the BIACORE technology as described in Example 1.1,and are shown in Tables 6 and 7, below. Their in vitro activities werefurther examined using other methods described in Example 1, and aresummarized in Table 8. Further characterization determined 37D10 and40B10 were identical rat mAbs.

TABLE 6 Biacore Kinetics of Anti-DLL4 Rat Hybridoma Antibodies Bindingto Human and Cynomolgus Monkey DLL4. Kinetics on Biacore huDLL4 ECDcynoDLL4 ECD k_(a) k_(d) K_(D) k_(a) k_(d) K_(D) Clone (M⁻¹s⁻¹) (s⁻¹)(nM) (M⁻¹s⁻¹) (s⁻¹) (nM) 38H12 4.0 × 10⁺⁵ 1.1 × 10⁻⁴ 0.3 2.6 × 10⁺⁵ 9.8× 10⁻⁵ 0.4 1A11 1.9 × 10⁺⁵ 1.1 × 10⁻³ 5.8 1.1 × 10⁺⁵ 9.3 × 10⁻⁴ 8.137D10 = 1.4 × 10⁺⁵ 7.0 × 10⁻² 484 4.0 × 10⁺⁴ Too <617 40B10 fast* 32C72.8 × 10⁺⁵ 7.0 × 10⁻⁶ 0.03 2.2 × 10⁺⁵ 6.3 × 10⁻⁶ 0.03 15D6 1.1 × 10⁺⁵1.0 × 10⁻³ 9.2 5.6 × 10⁺⁴ 8.3 × 10⁻⁴ 14.9 14A11 1.2 × 10⁺⁵ 1.2 × 10⁻³10.3 1.1 × 10⁺⁵ 1.2 × 10⁻³ 11.6 14G1 5.4 × 10⁺⁴ 3.9 × 10⁻⁴ 7.2 3.9 ×10⁺⁴ 4.1 × 10⁻⁴ 10.5 13E4 2.3 × 10⁺⁵ 3.9 × 10⁻⁴ 1.7 1.5 × 10⁺⁵ 4.2 ×10⁻⁴ 2.8 hu = human; cyno = cynomolgus monkey; *= falls out ofmeasurement range

TABLE 7 Biacore Kinetics of Anti-DLL4 Rat Hybridoma Antibodies BindingMurine and Rat DLL4. Kinetics on Biacore muDLL4 ECD ratDLL4 ECD k_(a)k_(d) K_(D) k_(a) k_(d) K_(D) Clone (M⁻¹s⁻¹) (s⁻¹) (nM) (M⁻¹s⁻¹) (s⁻¹)(nM) 38H12 N/B N/B N/B N/B N/B N/B 1A11 1.3 × 10⁺⁵ 2.9 × 10⁻³ 23 N/B N/BN/B 37D10 = 1.0 × 10⁺⁵ 1.35 × 10⁻²  135 N/D N/D N/D 40B10 32C7 4.8 ×10⁺⁴ 4.1 × 10⁻³ 88 N/D N/D N/D 15D6 7.4 × 10⁺⁴ 5.1 × 10⁻⁵ 0.7 N/B N/BN/B 14A11 7.0 × 10⁻⁴ 9.0 × 10⁻⁵ 1.3 N/B N/B N/B 14G1 N/B N/B N/B N/B N/BN/B 13E4 1.8 × 10⁺⁵ 1.9 × 10⁻⁴ 1.1 N/B N/B N/B N/B = no significantbinding; N/D = not determined, mu = mouse

TABLE 8 In Vitro Characterization of Rat Hybridoma Derived Anti-DLL4Antibodies. Functional Blockade Assays Notch Direct Binding AssaysCompetition Competition activation Binding ELISA FACS Inhibition ELISAFACS (IC₅₀, nM) (IC₅₀, nM) (IC₅₀, nM) (EC₅₀, nM) (EC₅₀, nM) DLL4 ECD/huNotch-1/ huDLL4 DLL4 ECD DLL4 Cells huNotch-1 DLL4 cells cell/NotchRat mAb hu mu hu mu hu mu hu mu reporter cells 1A11 0.06 0.11 34.8 0.80.78 0.79 8.4 0.8 2.9 38H12 0.06 — 1.2 — 3.63 — 1.7 — 0.5 37D10 = 0.120.14 2.3 0.7 5.39 3.36 5.5 3.8 — 40B10 32C7 0.08 0.15 3.1 0.8 — — — —Agonist 14G1 N/D N/D 8.6 >50 N/D N/D 4.2 — 6.1 14A11 N/D N/D >50 2.1 —19.8 205 1.6 2.7 15D6 N/D N/D 15 1.6 — — 25 1.5 3.2 13E4 N/D N/D 26 1.3— — 5.2 >200 — “—” = no activity or under detection limit of the assaysused; “N/D” = not determined; hu = human; cyno = cynomolgus monkey; mu =mouse.

Example 4. Deduction of Variable Region Protein Sequences of Anti-DLL4Rat Monoclonal Antibodies by DNA Cloning and Sequencing

Total RNA was extracted from hybridoma cell pellets using RNeasy minikit (Qiagen, catalog #74104) using the following protocol. 600 μl ofbuffer RLT were added to disrupt cells by pipetting up and down severaltimes. The cell lysate was homogenized by passing it 10 times through a20-gauge needle fitted to an RNase-free syringe. One volume of 70%ethanol was added to the homogenized lysate and mixed well by pipetting.Up to 700 μl at a time of the sample were added to an RNeasy spin columnand spun for 15 seconds at 10,000 rpm, discarding flow through. 700 μlof buffer RW1 were added to the column and spun for 15 seconds at 10,000rpm, discarding flow through. 500 μl of buffer RPE were added to washthe column membrane and spun for 15 seconds at 10,000 rpm, discardingflow through. The same step was repeated one more time, but the columnwas centrifuged for 2 minutes. Sample was then centrifuged for 1 minuteat 10,000 rpm to eliminate any carryover of buffer RPE. RNA was elutedwith 30 μl of RNase-free water by centrifuging for 1 minute at 10,000rpm. Subsequently, 2 μg of total RNA were used to synthesizefirst-strand cDNA using SuperScript First-Strand Synthesis System forRT-PCR (Invitrogen, catalog #11904-018) according tofollowing protocol:2 μg of RNA+2 μl dNTP+2 μl Oligo (dT)+DEPC-H₂O (to 20 μl) were incubatedat 65° C. for 5 minutes, then transferred to ice for at least 1 minute.The sample was then added to the following mixture: 4 μl of 10×RTbuffer+8 μl 25 mM MgCl₂+4 μl 0.1 M DTT+2 μl RNase OUT and incubated at42° C. for 2 minutes. Then, 2 μl of SuperScript II RT were added to thesample and incubated at 42° C. for 50 minutes. Sample was then incubatedat 70° C. for 15 minutes and chilled on ice. 2 μl of RNase H were thenadded and the sample was incubated at 37° C. for 20 minutes. cDNA wasthen used as template for PCR amplification of variable regions ofantibodies. PCR was performed using first-strand cDNA, primers fromMouse Ig-Primer Set (Novagen, catalog #69831-3) and Platinum Super MixHigh Fidelity (Invitrogen, catalog #12532-016). To amplify heavy chainvariable regions, PCR samples were assembled as follows: 22.5 μl PCRSuper Mix+0.25 μl reverse primer MuIgG V_(H)3′-2+1 μl cDNA+1.25 μl ofone the forward primers (VH-A, VH-B) or 0.5 μl of one of the forwardprimers (VH-C, VH-D, VH-E, VH-F). To amplify light chain variableregions PCR samples were assembled as follows: 22.5 μl PCR SuperMix+0.25 μl reverse primer MuIgKV_(L)-3′-1+1 μl cDNA+1.25 μl of one theforward primers (VL-A, VL-B) or 0.5 μl of one of the forward primers(VL-C, VL-D, VL-E, VL-F, VL-G).

For samples with primers VH-A, VH-B, VL-A and VL-B, the following PCRcycles were used (40-45 cycles, steps 2 through 4):

1—Denature 94° C. 2 min. 2—Denature 94° C. 30 sec. 3—Anneal 50° C. 30sec. 4—Extend 68° C. 1 min.

5—Final extension 68° C. 5 min.6—Cool 4° C. forever

For samples with primers VH-C through VH-F, and VL-C through VL-G, thefollowing PCR cycles were used (40-45 cycles, steps 2 through 4):

1—Denature 94° C. 2 min. 2—Denature 94° ° C. 30 sec. 3—Anneal 60° C. 30sec. 4—Extend 68° C. 1 min.

5—Final extension 68° C. 5 min.6—Cool 4° C. forever

PCR products were run on 1.2% agarose gel, and bands migrating at theexpected size (400-500 bp) were excised for DNA extraction. DNA waspurified using QIAquick Gel Extraction Kit (Qiagen, catalog #28704)according to the following protocol: gel slices were weighed. 3 volumesof buffer QG to 1 volume of gel were added to each gel slice. Sampleswere incubated at 50° C. for 10 minutes until gel slices were completelydissolved, mixing every 2-3 minutes. One gel volume of isopropanol wasthen added to each sample and mixed. Samples were then applied toQIAquick column and centrifuged for 1 minute at 13000 rpm. To wash, 750μl of buffer PE were added to samples and spun for 1 minute at 13000rpm. Columns were then centrifuged for an additional minute at 13,000rpm to completely remove residual ethanol. DNA was eluted by adding 30μl of H₂O to each column and by spinning 1 minute at 13,000 rpm.Purified PCR products were then sequenced to identify variable regionsequences (Table 9, below).

TABLE 9  VH and VL Amino Acid Sequences ofRat Anti-DLL4 Monoclonal Antibodies. SEQ ID Protein  NO region Sequence123456789012345678901234567890 157 VH 38H12EVQLVESGGGLVQPGRSLKLSCAASGFTFS NYGMYWIRQAPTKGLQWVAFISHGGGITYYRDSVKGRFT1SRDNAKSTLYLQMDSLRSED TATYHCAALNWELGIDYWGQGVMVTVSS VH 38H12Residues 31-35 NYGMY CDR-H1 of SEQ ID NO: 157 VH 38H12 Residues 50-66FISHGGGITYYRDSVKG CDR-H2 of SEQ ID NO: 157 VH 38H12 Residues 99-LNWELGIDY CDR-H3 107 of SEQ ID NO: 157 123456789012345678901234567890123456789012345678901234567890 158 VL 38H12DIQMTQSPASLSASLGETISIECRASEDIY SNLAWYQKKSGKSPQLLIYAANRLQDGVPSRFSGSGSGTQYSLKISGMQPEDEGDYFCLQ GSKFPLTFGSGTKLEIKR VL 38H12Residues 24-34 RASEDIYSNLA CDR-L1 of SEQ ID NO: 158 VL 38H12Residues 50-56 AANRLQD CDR-L2 of SEQ ID NO: 158 VL 38H12 Residues 89-97LQGSKFPLT CDR-L3 of SEQ ID NO: 158 123456789012345678901234567890 159VH 1A11 EVQLVESGGGLVQPGRSMKLSCAASGFTFR NFPMAWVRQAPTRGLEWVATISSSDGTTYYRDSVKGRFTISRDNAKSTLYLQVNSLRSED TATYYCSRGYYNSPFAYWGQGTLVTVSS123456789012345678901234567890 VH 1A11 Residues 31-35 NFPMA CDR-H1of SEQ ID NO: 159 VH 1A11 Residues 50-66 TISSSDGTTYYRDSVKG CDR-H2of SEQ ID NO: 159 VH 1A11 Residues 99- GYYNSPFAY CDR-H3 107 of SEQ IDNO: 159 123456789012345678901234567890 160 VL 1A11DIQMTQSPASLSASLGETVT1ECRASEDIY SNLAWYQQKPGNSPQLLIFDTNNLADGVPSRFSGSGSGTQSSLKINSLQSEDVASYFCQQ YNNYPPTFGGGTKLELKR VL 1A11 Residues 24-34RASEDIYSNLA CDR-L1 of SEQ ID NO: 160 VL 1A11 Residues 50-56 DTNNLADCDR-L2 of SEQ ID NO: 160 VL 1A11 Residues 89-97 QQYNNYPPT CDR-L3of SEQ ID NO: 160 123456789012345678901234567890123456789012345678901234567890 161 VH 37D10AVQLVESGGGLVQPKESLKISCAASGFTFS NAAMYWVRLAPGKGLEWVARIRTKPNNYATYYAESVKGRFTISRDDSKSMVYVQMDNLKT EDTAMYYCTAAPWRDSYAHVYWGQGVMVTV VH 37D10Residues 31-35 NAAMY CDR-H1 of SEQ ID NO: 161 VH 37D10 Residues 50-68R1RTKPNNYATYYAESVKG CDR-H2 of SEQ ID NO: 161 VH 37D10 Residues 101-APWRDSYAHVY CDR-H3 111 of SEQ ID NO: 161 123456789012345678901234567890162 VL 37D10 DIQMTQSPPVLSASVGDRVTLSCKASQNIHKNLDWYQQKHGDAPKLLIYYTDHLQTGVPS RFSGSGSATDYTLTISSLQPEDVATYYCYQYNGGPFTFGSGTKLEIKR 123456789012345678901234567890 VL37D10 Residues 24-34KASQNIHKNLD CDR-L1 of SEQ ID NO: 162 VL37D10 Residues 50-56 YTDHLQTCDR-L2 of SEQ ID NO: 162 VL37D10 Residues 89-97 YQYNGGPFT CDR-L3of SEQ ID NO: 162 123456789012345678901234567890 163 VH 32C7EVQLVESGGGLVQPGRSLKLSCLASGFPFS SVWMTWIRQAPGKGLEWIATITNSGASTYYSASVKGRFTISRDNVKSTLYLQMTSLGSED TATYYCTRVGTSFDYWGQGVMVTVSS VH 32C7Residues 31-35 SVWMT CDR-H1 of SEQ ID NO: 163 VH 32C7 Residues 50-66TITNSGASTYYSASVKG CDR-H2 of SEQ ID NO: 163123456789012345678901234567890 VH 32C7 Residues 99- VGTSFDY CDR-H3105 of SEQ ID NO: 163 123456789012345678901234567890 164 VL 32C7DIQMTQSPASLSASLGETVTIECRASDDIY NGLAWFQQKPGKSPQLLIYDANTLHTGVPSRFSGSGSGTQFSLKINSLQSEDVASYFCQQ FYDYPPYTFGAGTKLELKR VL 32C7Residues 24-34 RASDDIYNGLA CDR-L1 of SEQ ID NO: 164 VL 32C7Residues 50-56 DANTLHT CDR-L2 of SEQ ID NO: 164 VL 32C7 Residues 89-98QQFYDYPPYT CDR-L3 of SEQ ID NO: 164 123456789012345678901234567890123456789012345678901234567890 165 VH 14G1EVQLQQSGAELAKPGSSVKISCKASGYTFT NYDISWIKQTNGQGLEYLGYINTGSGGIYSNEKFKGKATLTVDKSSNTAFMQLSSLTPED TAVYYCVREGNNFDHWGQGVK VTVSS VH 14G1Residues 31-35 NYDIS CDR-H1 of SEQ ID NO: 165 VH 14G1 Residues 50-66YINTGSGG1YSNEKFKG CDR-H2 of SEQ ID NO: 165 VH 14G1 Residues 99- EGNNFDHCDR-H3 105 of SEQ ID NO: 165 123456789012345678901234567890 166 VL 14G1DTVMTQSPASMSTSVGERVTVNCKASQSVG TIVAWFQQKPGQSPKRLIYLATYRHTGVPDRFIGSGFGRDFTLTISNVEAEDLAVYYCLQ YGSRPFTFGAGTKLEIKR123456789012345678901234567890 VL 14G1 Residues 24-34 KASQSVGTIVA CDR-L1of SEQ ID NO: 166 VL 14G1 Residues 50-56 LATYRHT CDR-L2 of SEQ IDNO: 166 VL 14G1 Residues 89-97 LQYGSRPFT CDR-L3 of SEQ ID NO: 166123456789012345678901234567890 167 VH 14A11EVQLQQSGPELAKPGSSVKISCKASGYTFT NSYISWIKQTTGQGLEYVGYINTGSGGADYNEKFKGKATLTVDKSSRTAFMQLSSLTPGD SAVYYCAKSILLGSTCYFDYWGQGVLVTVS VH 14A11Residues 31-35 SNSYIS CDR-H1 of SEQ ID NO: 167 VH 14A11 Residues 50-66YINTGSGGADYNEKFKG CDR-H2 of SEQ ID NO: 167123456789012345678901234567890 VH 14 A11 Residues 99- SILLGSTCYFDYCDR-H3 110 of SEQ ID NO: 167 123456789012345678901234567890 168 VL 14A11NTVLTQSPALAVSLGQRVTISCKASRSVSS PMYSYIYWYQQKPGQQPKLLIYRASTLASGVPARFSGSGSGTDFTLNIDPVEADDIATYF CQQSWSDPFTFGSGTKLEIKR VL 14A11Residues 23-37 KASRSVSSPMYSYIY CDR-L1 of SEQ ID NO: 168 VL 14A11Residues 53-59 RASTLAS CDR-L2 of SEQ ID NO: 168 VL 14A11 Residues 92-QQSWSDPFT CDR-L3 100 of SEQ ID NO: 168 123456789012345678901234567890123456789012345678901234567890 169 VH 15D6EVQLQQSGPELAKPGSSVKISCKASGYTFT SSYISWIKQTTGQGLEYIGYINTGSGGTDYNEKFKDKATLTVDKSSRTVFMQLSSLTPGD SAVYYCAKSILLGSTYYLDYWGQGVMVTVS S VH 15D6Residues 31-35 SSYIS CDR-H1 ofSEQ ID NO: 169 VH 15D6 Residues 50-66YINTGSGGTDYNEKFKD CDR-H2 of SEQ ID NO: 169 VH 15D6 Residues 99-SILLGSTYYLDY CDR-H3 110 of SEQ ID NO: 169 123456789012345678901234567890170 VL 15D6 DTVLTQSPALAVSLGQRVTISCKASRSLSSPMYSYIYWYQQKLGQQPRLLIYRASTLASG VPARFSGSGSGTDFTLNIDPVEADDIATYFCQQSWSDPFTFGSGTKLEIKR 123456789012345678901234567890 VL 15D6Residues 23-37 KASRSLSSPMYSYIY CDR-L1 ofSEQ ID NO: 170 VL 15D6Residues 53-59 RASTLAS CDR-L2 ofSEQ ID NO: 170 VL 15D6 Residues 92-QQSWSDPFT CDR-L3 100 of SEQ ID NO: 170

Example 5. Generation of Chimeric Antibodies

The variable domains of the heavy and light chain of the anti-DLL4 ratmAbs (Table 9, above) were cloned in-frame to mutant human IgG1 (L234,235A) heavy-chain and kappa light-chain constant regions, respectively.The activities of the resulting chimeric antibodies were confirmed inFACS-based binding and competition assays (Table 10, below), and werecomparable to their parental rat mAbs.

TABLE 10 FACS-Based Binding and Neutralizing Activity of RecombinantChimeric Antibodies Containing the Variable Domains of the Anti-DLL4 RatmAbs. FACS binding Competition FACS (EC₅₀ nM) (IC₅₀ nM) DLL4 cellshuNotch-1/DLL4 cells Human Mouse Human Mouse Chimera of DLL4 DLL4 DLL4DLL4 1A11 19.59 0.74  2.338 0.682 38H12 1.468 N/D 1.443 N/D 32C7 3.7064.114 ND ND 37D10 2.32 0.99  5.951 4.395 14G1 0.994 N/D 4.11  N/D 14A111.613 2.139 4.025 1.391 15D6 1.715 1.817 10.48  1.49  N/D = notdetermined.

Example 6. Humanization of Anti-DLL4 Rat Monoclonal Antibody 1A11

1A11 rat anti-DLL4 antibody (Table 9, above) was humanized. Humanizedvariant amino acid sequences VH.1, VH.1a, VH.1b, VH.2a, VL.1, VL.1a,VL.1b, and VL.2a (Table 11, below) were converted to DNA sequence basedon the most homologous human germlines and synthesized. For the heavychain variants, human germline heavy chain acceptor sequences VH3-7 FR1,VH3-7 FR2, VH3-7 FR 3, and JH4 FR4 were used (see, Table 3, above). Forlight chain variants VL.1, VL.1a, and VL.1b, human germline light chainacceptor sequences O2 FR1, O02 FR2, O2 FR3, and JK2 FR4 were used (see,Table 3, above). For light chain variant VL.2a, human germline lightchain acceptor sequences L2 FR1, L2 FR2, L2 FR3, and JK2 FR4 were used(see, Table 4, above). Individual constructs were sequence verified tocheck for accuracy. Positive variants were then inoculated into 250 mlsLuria broth plus ampicillin and cultured overnight at 37° C. DNA wasextracted from variant cultures using the Qiagen Hi speed maxi prep kit(12662).

TABLE 11  VH and VL Amino Acid Sequences of Humanized Versionsof Rat Anti-DLL4 Monoclonal Antibody 1A11. SEQ ID Protein  NO: regionSequence 123456789012345678901234567890 171 VH.1 1A11EVQLVESGGGLVQPGGSLRLSCAASGFTFS NFPMAWVRQAPGKGLEWVATISSSDGTTYYRDSVKGRFTISRDNAKNSLYLQMNSLRAED TAVYYCARGYYNSPFAYWGQGTLVTVSS123456789012345678901234567890 VH.1 1A11 Residues NFPMA CDR-H1 31-35 ofSEQ ID NO: 171 VH.1 1A11 Residues TISSSDGTTYYRDSVKG CDR-H2 50-66 ofSEQ ID NO: 171 VH.1 1A11 Residues GYYNSPFAY CDR-H3 99-107 of SEQ IDNO: 171 123456789012345678901234567890 172 VH.1a 1A11EVQLVESGGGLVQPGGSLRLSCAASGFTFS NFPMAWVRQAPGKGLEWVATISSSDGTTYYRDSVKGRFTISRDNAKSSLYLQMNSLRAED TAVYYCSRGYYNSPFAYWGQGTLVTVSS VH.1a 1A11Residues NFPMA CDR-H1 31-35 of SEQ ID NO: 172 VH.1a 1A11 ResiduesTISSSDGTTYYRDSVKG CDR-H2 50-66 of SEQ ID NO: 172123456789012345678901234567890 VH.1a 1A11 Residues GYYNSPFAY CDR-H399-107 of SEQ ID NO: 172 173 VH.1b 1A11 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNFPMAWVRQAPGKGLEWVATISSSDGTTYY RDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCSRGYYNSPFAYWGQGTLVTVSS VH.1b 1A11 Residues NFPMA CDR-HI 31-35 ofSEQ ID NO: 173 VH.1b 1A11 Residues TISSSDGTTYYRDSVKG CDR-H2 50-66 ofSEQ ID NO: 173 VH.1b 1A11 Residues GYYNSPFAY CDR-H3 99-107 of SEQIDNO: 173 123456789012345678901234567890 174 VH.2a 1A11EVQLVESGGGLVQPGGSLRLSCAASGFTFS NFPMAWVRQAPGKGLEWVATISSSDGTTYYRDSVKGRFTISRDNSKSTLYLQMNSLRAED TAVYYCSRGYYNSPFAYWGQGTLVTVSS VH.2a 1A11Residues NFPMA CDR-H1 31-35 of SEQ ID NO: 174 VH.2a 1A11 ResiduesTISSSDGTTYYRDSVKG CDR-H2 50-66 of SEQ ID NO: 174 VH.2a 1A11 ResiduesGYYNSPFAY CDR-H3 99-107 of SEQ ID NO: 174 123456789012345678901234567890175 VL.1 1A11 DIQMTQSPSSLSASVGDRVTITCRASEDIYSNLAWYQQKPGKAPKLLIYDTNNLADGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPPTFGQGTKLEIKRRASEDIYSNLA VL.1 1A11 Residues CDR-L1 24-34 of SEQ IDNO: 175 VL.1 1All Residues DTNNLAD CDR-L2 50-56 of SEQID NO: 175VL.1 1A11 Residues QQYNNYPPT CDR-L3 89-97 of SEQ ID NO: 175123456789012345678901234567890 176 VL. 1a 1A11DIQMTQSPSSLSASVGDRVTITCRASED1Y SNLAWYQQKPGKSPKLLIFDTNNLADGVPSRFSGSGSGTDSTLTISSLQPEDFATYFCQQ YNNYPPTFGQGTKLEIKR VL. 1a 1A11 ResiduesRASEDIYSNLA CDR-Ll 24-34 of SEQ ID NO: 176 VL.1a 1A11 Residues DTNNLADCDR-L2 50-56 of SEQ ID NO: 176 VL.1a 1A11 Residues QQYNNYPPT CDR-L389-97 of SEQ ID NO: 176 123456789012345678901234567890 177 VL. 1b 1A11DIQMTQSPSSLSASVGDRVTITCRASEDIY SNLAWYQQKPGKAPKLLIFDTNNLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ YNNYPPTFGQGTKLEIKR VL. 1b 1A11 ResiduesRASEDIYSNLA CDR-L1 24-34 of SEQ ID NO: 177 VL. 1b 1A11 Residues DTNNLADCDR-L2 50-56 of SEQ ID NO: 177 VL. 1b 1A11 Residues QQYNNYPPT CDR-L389-97 of SEQ ID NO: 177 123456789012345678901234567890 178 VL.2A 1A11EIVMTQSPATLSVSPGERATLSCRASEDIY SNLAWYQQKPGQSPRLLIFDTNNLADGVPARFSGSGSGTESTLTISSLQSEDFAVYFCQQ YNNYPPTFGQGTKLEIKR VL.2a 1A11 ResiduesRASEDIYSNLA CDR-L1 24-34 of SEQ ID NO: 178 VL.2a 1A11 Residues DTNNLADCDR-L2 50-56 of SEQID NO: 178 VL.2a 1A11 Residues QQYNNYPPT CDR-L389-97 of SEQ ID NO: 178

Humanized antibodies were generated by combining each heavy chainvariant with each light chain variant for a total of 16 variants (Table12, below). Variants 1-4 each contained VH.1 paired with each VLvariant: VL.1: 0 back mutations, VL.1a: 4 back mutations, VL.1b: 1 backmutation, and VL.2a: 5 back mutations. Variants 5-8 each contained VH.1apaired with each VL variant: VL.1: 2 back mutations, VL.1a: 6 backmutations, VL.1b:3 back mutations, and VL.2a: 7 back mutations. Variants9-12 each contained VH.1b paired with each VL variant: VL.1: 1 backmutation, VL.1a: 5 back mutations, VL.1b: 2 back mutations, and VL.2a: 6back mutations. Variants 13-16 each contained VH.2a paired with each VLvariant: VL.1: 3 back mutations, VL.1a: 7 back mutations, VL.1b: 4 backmutations, VL.2a: 8 back mutations.

TABLE 12 Summary of Humanized 1A11 Antibodies Generated and BackMutations. Back Mutations in VH/VL Variable Regions^(†) Name Combination(VH/VL) h1A11.1 VH.1/VL.1 0/0 h1A11.2 VH.1/VL.1a 0/A43S, Y49F, F71S,Y87F h1A11.3 VH.1/VL.1b 0/Y49F h1A11.4 VH.1/VL.2a 0/A43S, Y49F, I58V,F71S, Y87F h1A11.5 VH.1a/VL.1 N76S, A93S/0 h1A11.6 VH.1a/VL.1a N76S,A93S/A43S, Y49F, F71S, Y87F h1A11.7 VH.1a/VL.1b N76S, A93S/Y49F h1A11.8VH.1a/VL.2a N76S, A93S/A43S, Y49F, I58V, F71S, Y87F h1A11.9 VH.1b/VL.1A93S/0 h1A11.10 VH.1b/VL.1a A93S/A43S, Y49F, F71S, Y87F h1A11.11VH.1b/VL.1b A93S/Y49F h1A11.12 VH.1b/VL.2a A93S/A43S, Y49F, 158V, F71S,Y87F h1A11.13 VH.2a/VL.1 S49A, N76S, A93S/0 h1A11.14 VH.2a/VL.1a S49A,N76S, A93S,/A43S, Y49F, F71S, Y87F h1A11.15 VH.2a/VL.1b S49A, N76S,A93S/ Y49F h1A11.16 VH.2a/VL.2a S49A, N76S, A93S/A43S, Y49F, I58V, F71S,Y87F ^(†)Kabat numbering used.

All 16 variants were transiently transfected into 50 mls of HEK 293 6esuspension cell cultures in a ratio of 60% to 40% light to heavy chainconstruct. 1 mg/ml PEI was used to transfect the cells. Cellsupernatants were harvested after six days in shaking flasks, spun downto pellet cells, and filtered through 0.22 μm filters to separate IgGfrom culture contaminates. Variant binding to human DLL4 was initiallyassessed through a capture binding ELISA (Example 1.2), which utilized agoat anti human-Fc capture antibody (Jackson ImmunoResearch,109-005-008) to capture IgG within filtered 293 6e cell supernatants(Table 13, below). All 16 variants had very comparable affinities andwere purified for further characterization.

All 16 variants (h1A11.1-h1A11.16) were batch purified by adding 1supernatant volume of protein A IgG binding buffer (Thermo Scientific21001) and 1 ml of rProteinA sepharose fast flow beads (GE Healthcare,17-1279-04). Supernatants, with beads and buffer added, were rockedovernight at 4° C., and the day after beads were collected by gravityover poly prep chromatography columns (Bio Rad, 731-1550). Oncesupernatants had passed through the columns the beads were washed with10 column volumes of binding buffer, and IgG was eluted with ImmunopureIgG elution buffer (Pierce, 185 1520) and collected in 1 ml aliquots.Fractions containing IgG were pooled and dialyzed in PBS overnight at 4°C.

Purified variants were further characterized for their affinities forhuman, murine and cynomolgus DLL4 by binding ELISA (Example 1.2, Method2), by Biacore (Example 1.1), and by Flow Cytometry (FACS). All 16variants showed comparable affinities to the parent recombinant antibody1A11 in all the three assays (Table 13). The humanized variants werethen tested for their functionality with human and murine DLL4 bycompetition ELISA (Example 1.3) and by Notch reporter assay (Example1.6). All 16 variants showed comparable potencies to the parentrecombinant antibody 1A11 in both assays (Table 14, below).

TABLE 13 Summary of Binding Activities of Humanized 1A11 mAbs. BindingData Human DLL4 Mouse DLL4 Cyno DLL4 Binding Binding Binding BindingBinding ELISA Biacore FACS ELISA Biacore FACS ELISA mAb (EC50, nM) (KD,M) (EC50, nM) (EC50, nM) (KD, M) (EC50, nM) (EC50, nM) h1A11-1 0.08 1.5× 10⁻⁸ 3.13 0.09 3.8 × 10⁻⁸ 0.21 0.13 h1A11-2 0.08 1.0 × 10⁻⁸ 2.85 0.09 1.9 × 10⁻⁸* 0.21 0.13 h1A11-3 0.07 1.5 × 10⁻⁸ 3.51 0.08 3.7 × 10⁻⁸ 0.240.16 h1A11-4 0.08 1.6 × 10⁻⁸ 3.61 0.09 3.9 × 10⁻⁸ 0.22 0.17 h1A11-5 0.070.96 × 10⁻⁸  3.61 0.08 2.2 × 10⁻⁸ 0.21 0.16 h1A11-6 0.08 1.13 × 10⁻⁸ 3.74 0.10 3.1 × 10⁻⁸ 0.15 0.13 h1A11-7 0.06 1.3 × 10⁻⁸ 3.71 0.09 3.5 ×10⁻⁸ 0.18 0.14 h1A11-8 0.06 1.1 × 10⁻⁸ 3.34 0.09 2.4 × 10⁻⁸ 0.18 0.17h1A11-9 0.07 1.3 × 10⁻⁸ 3.28 0.09 3.4 × 10⁻⁸ 0.18 0.15 h1A11-10 0.06 1.3× 10⁻⁸ nt 0.09 3.5 × 10⁻⁸ nt 0.15 h1A11-11 0.07 1.2 × 10⁻⁸ nt 0.09 2.7 ×10⁻⁸ nt 0.15 h1A11-12 0.06 1.4 × 10⁻⁸ nt 0.08 3.5 × 10⁻⁸ nt 0.16h1A11-13 0.06 1.4 × 10⁻⁸ nt 0.09 3.7 × 10⁻⁸ nt 0.16 h1A11-14 0.055 1.3 ×10⁻⁸ nt 0.07 2.8 × 10⁻⁸ nt 0.14 h1A11-15 0.07 1.4 × 10⁻⁸ nt 0.09 3.7 ×10⁻⁸ nt 0.13 h1A11-16 0.06 1.5 × 10⁻⁸ nt 0.09 3.9 × 10⁻⁸ nt 0.12 1A110.2 0.9 × 10⁻⁸ 6.29 0.3 2.4 × 10⁻⁸ 0.53 0.33 (chimeric) 1A11 (rat 0.6 ×10⁻⁸ mAb) nt = not tested

TABLE 14 Summary of In Vitro Functional Potency of Humanized 1A11 mAbs.Functional Data Human DLL4 Mouse DLL4 Competition Reporter CompetitionELISA Assay ELISA mAb (IC50, nM) (EC50, nM) (IC50, nM) h1A11-1 0.4 2.531.3 h1A11-2 0.3 3.92 0.9 h1A11-3 0.2 2.53 0.9 h1A11-4 0.3 3.28 0.9h1A11-5 0.3 3.8 0.8 h1A11-6 0.4 1.45 0.8 h1A11-7 0.5 4.84 0.9 h1A11-80.35 4.24 0.9 h1A11-9 0.3 3.18 0.9 h1A11-10 0.35 5.88 0.9 h1A11-11 0.43.73 0.8 h1A11-12 0.4 2.89 0.9 h1A11-13 0.3 10.42 1 h1A11-14 0.25 4.10.7 h1A11-15 0.2 5.4 0.7 h1A11-16 0.3 2.61 0.7 1A11 (chimera) 1.8 5.983.5 1A11 (rat 0.5 1.1 hybridoma mAb)

Additional Designs for Humanizing Anti-DLL4 1A11 Antibodies.

Additional V_(H) and VL designs for humanizing anti-DLL4 rat monoclonalantibody 1A11 are shown in the table below.

TABLE 15 Additional VH and VL Designs for Humanizing 1A11 Antibodies.Acceptor Framework VH or VL Design Sequence Back Mutations^(†) h1A11VH.2VH3 CONSENSUS + JH4 0 h1A11VH.2b VH3 CONSENSUS + JH4 S49A, A93Sh1A11VH.3 VH1-46 + JH4 0 (with Q1E to prevent N- terminal pyroglutamateformation) h1A11VH.3b VH1-46 + JH4 Y27F, M48V, G49A, (Q1E) A93Sh1A11VH.3c VH1-46 + JH4 Y27F, M48V, G49A, (Q1E) V67F, M69I, T73N, V78L,A93S, and TV5I (to avoid undesirable N- glycosylation signal) h1A11VH.3dVH1-46 + JH4 Y27F, M48V, G49A, (Q1E) V67F, M69I, V78L, A93S (T73Nomitted to avoid undesirable N- glycosylation signal) h1A11VL.1c O2 +JK2 Y49F, F71S h1A11VL.2 3-15/L2 + JK2 0 h1A11VL.2b 3-15/L2 + JK2 Y49Fh1A11VL.2c 3-15/L2 + JK2 Y49F, F71S ^(†)Kabat numbering used.

Example 7. Humanization of Anti-DLL4 Rat mAb 38H12

The 38H12 rat anti-DLL4 antibody (Table 9, above) was humanized.Humanized variant amino acid sequences VH.1, VH.1a, VH.1b, VH.2a, VL.1,VL.1a, VL.1b, and VL.2a (Table 16, below) were converted to DNAsequence, based on the most homologous human germlines, and synthesized.Human germline heavy chain acceptor sequences VH3-30 FR1, VH3-30 FR2,VH3-30 FR3, and JH3 FR4 (see, Table 3) were used for generating thehumanized heavy chain variants shown in Table 16. Human germline lightchain acceptor sequences O2 FR1, O2 FR2, O2 FR3, and JK2 FR4 (see, Table4) were used for generating the humanized light chain variants shown inTable 16. Individual constructs were sequence verified to check foraccuracy. Positive variants were then inoculated into 150 mls Luriabroth plus ampicillin and cultured overnight at 37° C. DNA was extractedfrom variant cultures using the Qiagen Hi speed maxi prep kit (12662).

TABLE 16 VH and VL Amino Acid Sequences of HumanizedRat Anti-DLL4 Monoclonal Antibody 38H12. SEQ ID Protein NO: regionSequence 123456789012345678901234567890 179 VH.1 38H12EVQLVESGGGVVQPGRSLRLSC AASGFTFSNYGMYWVRQAPGKG LEWVAFISHGGGITYYRDSVKGRFTISRDNSKNTLYLQMNSLRA EDTAVYYCARLNWELGIDYWGQ GTMVTVSS VH.1 38H12Residues 31-35 of NYGMY CDR-H1 SEQ ID NO: 179 VH.1 38H12Residues 50-66 of FISHGGGITYYRDSVKG CDR-H2 SEQ ID NO: 179 VH.1 38H12Residues 99-107 LNWELGIDY CDR-H3 of SEQ ID NO: 179123456789012345678901234567890 180 VH.1a 38H12 EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMYWIRQAPGKG LEWVAFISHGGGITYYRDSVKGR FTISRDNSKSTLYLQMNSLRAEDTAVYHCAALNWELGIDYWGQG TMVTVSS VH.1a 38H12 Residues 31-35 of NYGMY CDR-H1SEQ ID NO: 180 123456789012345678901234567890 VH.1a 38H12Residues 50-66 of FISHGGGITYYRDSVKG CDR-H2 SEQ ID NO: 180 VH.1a 38H12Residues 99-107 of LNWELGIDY CDR-H3 SEQ ID NO: 180123456789012345678901234567890 181 VH.1b 38H12 EVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMYWVRQAPGKGLEWV AFISHGGGITYYRDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC AALNWELGIDYWGQGTMVTVSS VH.1b 38H12Residues 31-35 of NYGMY CDR-H1 SEQ ID NO: 181 VH.1b 38H12Residues 50-66 of FISHGGGITYYRDSVKG CDR-H2 SEQ ID NO: 181 VH.1b 38H12Residues 99-107 of LNWELGIDY CDR-H3 SEQ ID NO: 181123456789012345678901234567890 182 VH.2a 38H12 EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYGMYWIRQAPGKG LEWVAFISHGGGITYYRDSVKG RFTISRDNSKSTLYLQMNSLRAEDTAVYHCAALNWELGIDYWGQ GTMVTVSS VH.2a 38H12 Residues 31-35 of NYGMYCDR-H1 SEQ ID NO: 182 VH.2a 38H12 Residues 50-66 of FISHGGGITYYRDSVKGCDR-H2 SEQ ID NO: 182 VH.2a 38H12 Residues 99-107 of LNWELGIDY CDR-H3SEQ ID NO: 182 123456789012345678901234567890 183 VL.1 38H12DIQMTQSPSSLSASVGDRVTITC RASEDIYSNLAWYQQKPGKAPKL LIYAANRLQDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQGS KFPLTFGQGTKLEIKR VL.1 38H12 Residues 24-34 ofRASEDIYSNLA CDR-L1 SEQ ID NO: 183 VL.1 38H12 Residues 50-56 of AANRLQDCDR-L2 SEQ ID NO: 183 VL.1 38H12 Residues 89-97 of LQGSKFPLT CDR-L3SEQ ID NO: 183 123456789012345678901234567890 184 VL. 1a 38H12DIQMTQSPSSLSASVGDRVTITC RASEDIYSNLAWYQKKPGKSPKL LIYAANRLQDGVPSRFSGSGSGTDYTLTISSLQPEDFATYFCLQGS KFPLTFGQGTKLEIKR VL. 1a 38H12 Residues 24-34 ofRASEDIYSNLA CDR-L1 SEQ ID NO: 184 VL. 1a 38H12 Residues 50-56 of AANRLQDCDR-L2 SEQ ID NO: 184 VL. 1a 38H12 Residues 89-97 of LQGSKFPLT CDR-L3SEQ ID NO: 184 123456789012345678901234567890 185 VL. 1b 38H12DIQMTQSPSSLSASVGDRVTITC RASEDIYSNLAWYQQKPGKAPKL LIYAANRLQDGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCLQGS KFPLTFGQGTKLEIKR VL. 1b 38h12 Residues 24-34 ofRASEDIYSNLA CDR-L1 SEQ ID NO: 185 VL. 1b 38H12 Residues 50-56 of AANRLQDCDR-L2 SEQ ID NO: 185 VL. 1b 38H12 Residues 89-97 of LQGSKFPLT CDR-L3SEQ ID NO: 185 123456789012345678901234567890 186 VL.2a 38H12EIVMTQSPATLSVSPGERATLSC RASEDIYSNLAWYQKKPGQSPRL LIYAANRLQDGVPARFSGSGSGTEYTLTISSLQSEDFAVYFCLQGS KFPLTFGQGTKLEIKR VL.2a 38H12 Residues 24-34 ofRASEDIYSNLA CDR-L1 SEQ ID NO: 186 VL.2a 38H12 Residues 50-56 of AANRLQDCDR-L2 SEQ ID NO: 186 VL.2a 38H12 Residues 89-97 of LQGSKFPLT CDR-L3SEQ ID NO: 186

Humanized antibodies were generated by combining each heavy chainvariant with each light chain variant for a total of 16 variants (Table17, below). Variants 1-4 each contained VH.1 paired with each VLvariant: VL.1: 0 back mutations, VL.1a: 4 back mutations, VL.1b: 1 backmutation, and VL.2a: 5 back mutations. Variants 5-8 each contained VH.1apaired with each VL variant: VL.1: 4 back mutations, VL.1a: 8 backmutations, VL.1b: 5 back mutations, and VL.2a: 9 back mutations.Variants 9-12 each contained VH.1b paired with each VL variant: VL.1: 1back mutation, VL.1a: 5 back mutations, VL.1b: 2 back mutations, andVL.2a: 5 back mutations. Variants 13-16 each contained VH.2a paired witheach VL variant: VL.1: 5 back mutations, VL.1a: 9 back mutations, VL.1b:6 back mutations, VL.2a:10 back mutations.

TABLE 17 Summary of Humanized 38H12 Antibodies Generated and BackMutations. Back Mutations in VH/VL Variable Regions^(†) Name Combination(VH/VL) h38H12.1 VH.1/VL.1 0/0 (Q1E in VH to prevent N-terminalpyroglutamate formation) h38H12.2 VH.1/VL.1a 0/Q38K, A43S, F71Y, Y87F(Q1E in VH as noted above) h38H12.3 VH.1/VL.1b 0/F71Y (Q1E in VH asnoted above) h38H12.4 VH.1/VL.2a 0/Q38K, A43S, I58V, F71Y, Y87F (Q1E inVH as noted above) h38H12.5 VH.1a/VL.1 V37I, N76S, Y91H, R94A/0 h38H12.6VH.1a/VL.1a V37I, N76S, Y91H, R94A/Q38K, A43S, F71Y, Y87F h38H12.7VH.1a/VL.1b V37I, N76S, Y91H, R94A/F71Y h38H12.8 VH.1a/VL.2a V37I, N76S,Y91H, R94A/Q38K, A43S, I58V, F71Y, Y87F h38H12.9 VH.1b/VL.1 R94A/0h38H12.10 VH.1b/VL.1a R94A/Q38K, A43S, F71Y, Y87F h38H12.11 VH.1b/VL.1bR94A/F71Y h38H12.12 VH.1b/VL.2a R94A/Q38K, A43S, I58V, F71Y, Y87Fh38H12.13 VH.2a/VL.1 V37I, S49A, N76S, Y91H, R94A/0 h38H12.14VH.2a/VL.1a V37I, S49A, N76S, Y91H, R94A/Q38K, A43S, F71Y, Y87Fh38H12.15 VH.2a/VL.1b V37I, S49A, N76S, Y91H, R94A/F71Y h38H12.16VH.2a/VL.2a V37I, S49A, N76S, Y91H, R94A/Q38K, A43S, I58V, F71Y, Y87F^(†)Kabat numbering used.

All 16 variants were transiently transfected into 50 mls of HEK 293 6esuspension cell cultures in a ratio of 60% to 40% light to heavy chainconstruct. 1 mg/ml PEI was used to transfect the cells. Cellsupernatants were harvested after seven days in shaking flasks, spundown to pellet cells, and filtered through 0.22 μm filters to separateIgG from culture contaminates. Variant binding to human DLL4 wasinitially assessed through a capture binding ELISA (Example 1.2), whichutilized a goat anti human-Fc capture antibody (Jackson immuno research,109-005-008) to capture IgG within filtered 293 6e cell supernatants(see, ELISA binding EC50, Table 18, below). Variants containing VH.1exhibited the lowest binding affinities as compared to the othervariants and were considered out of the screening. VH.1 is CDR-graftedwith noframework back mutations.

Good binders (h38H12.5-h38H12.16) were then batch purified by adding 1supernatant volume of protein A IgG binding buffer (Thermo Scientific21001) and 800 μl of rProteinA sepharose fast flow beads (GE Healthcare,17-1279-04). Supernatants, with beads and buffer added, were stirred atroom temperature for 4 hours, and beads were collected by gravity overpoly prep chromatography columns (Bio Rad, 731-1550). Once supernatantshad passed through the columns the beads were washed with 10 mls bindingbuffer and IgG was eluted with Immunopure IgG elution buffer (Pierce,185 1520) and collected in 1 ml aliquots neutralized with 100 μl 1MTris, pH 8.

Purified variants were further characterized in human Notch-1competition ELISAs (Example 1.4), which used a format of plating Notch-1Fc onto ELISA plates and pre-incubating biotinylated human DLL4 plustitrated antibody. Signal was assessed by free biotinylated DLL4 bindingto Notch-1 Fc. Strong binders inhibited signal at low antibodyconcentration. h38H12.5 through h38H12.7 exhibited lower competitionpotencies as compared to the other variants (see, Notch CompetitionELISA EC₅₀, Table 18, below).

The good binder variants, as determined by binding ELISA, were assessedby Biacore (Example 1.1) concurrently with cell based assay screens. TheK_(D) for human DLL4 was similar for all variants (see, Biacore, K_(D)in Table 18, below). Variants were screened in cell-based assays thatexamined direct binding to human DLL4 (Example 1.3; FACS binding EC₅₀ inTable 18, below) and inhibition of Notch-1 signaling (Example 1.6; NotchReporter Assay EC₅₀ in Table 18, below).

TABLE 18 Summary of In Vitro Activities Against Human DLL4 of Humanized38H12 mAbs. Notch Notch ELISA Competition FACS Reporter binding ELISAbinding Assay EC₅₀ Biacore, Name EC₅₀ (nM) EC₅₀ (nM) EC₅₀ (nM) (nM)K_(D) (nM) h38H12.1 21.5 17.84 h38H12.2 26.88 h38H12.3 5.57 h38H12.420.65 h38H12.5 0.2015 14.81 77.15 7.307 0.401 h38H12.6 0.1584 26.9612.29 6.317 0.434 h38H12.7 0.1798 12.49 19.06 2.598 0.34 h38H12.8 0.19728.315 20.02 5.557 0.397 h38H12.9 0.1155 4.158 3.71 1.436 0.986 h38H12.100.1226 3.902 2.489 0.7861 0.578 h38H12.11 0.1264 3.8 2.477 0.6572 0.554h38H12.12 0.1651 3.228 1.478 1.062 0.819 h38H12.13 0.1534 5.287 2.5560.7943 0.507 h38H12.14 0.146 5.839 1.04 1.014 0.303 h38H12.15 0.09045.714 2.369 0.837 0.355 h38H12.16 0.1696 3.766 2.914 1.185 0.392

Additional Designs for Humanizing Anti-DLL4 38H12 Antibodies.

Additional VH and VL designs for humanizing anti-DLL4 rat monoclonalantibody 38H12 are shown in the table below.

TABLE 19 Additional VH and VL Designs for Humanizing 38H12 Antibodies.Germline Acceptor VH or VL Design Framework Sequences Back Mutations^(†)h38H12VH.2 VH3 CONSENSUS + JH3 0 h38H12VH.2b VH3 CONSENSUS + JH3 S49A,R94A h38H12VH.3 VH1-46 + JH3 0 (Q1E in VH to prevent N-terminalpyroglutamate formation) h38H12VH.3b VH1-46 + JH3 Y27F, M48V, G49A, R94A(Q1E in VH as noted above) h38H12VH.3c VH1-46 + JH3 Y27F, Y37I, M48V,G49A, V67F, M69I, T73N, V78L, Y91H, R94A (T75I to eliminate undesirableN- glycosylation signal, Q1E in VH as noted above) h38H12VH.3d VH1-46 +JH3 Y27F, Y37I, M48V, G49A, V67F, M69I, V78L, Y91H, R94A (Q1E in VH asnoted above) h38H12VL.2 3-15/L2 + JK2 0 h38H12VL.2b 3-15/L2 + JK2 F71Y^(†)Kabat numbering used.

Example 8. Affinity Maturation of h1A11.1

Humanized antibody h1A11.1 was used as a template for affinitymaturation. A description of the design of the library is providedbelow. Numbering of variable region sequences of monoclonal antibodieswas annotated with Kabat numbering (as described above; or see worldwidewebsite bioinf org.uk/abs/#kabatnum) and was used in generating thelibraries described below.

Three libraries were made as described below.H1+H2 library (doping: 76080808):

Doped 11 residues at 30, 31, 32, 35, 50, 52, 52a, 55, 56, 57, and 58.

Toggle between germline and h1A11 sequence at position 76(V/I).

A 10⁹ library sampled mutants with 4 or fewer mutated residues at least3.7 times.

The majority of the library with mutants carrying 4 to 6 residues wasmutated by doping.

H3 Library (Doping: 70101010)

Doped 8 residues @ 95, 96, 97, 98, 99, 100, 100a, and 102.

Toggle between germline and h1A11 sequence at position 93(A/S) and 101(D/A).

A 10⁹ library sampled mutants with 4 or fewer mutated residues at least4.7 times.

The majority of the library with mutants carrying 4 to 5 residues wasmutated by doping.

LC Library (Doping: 70101010):

Doped 9 residues at 28, 30, 31, 50, 53, 92, 93, 94, and 96.

Toggle between germline and h1A11 sequence at 7 positions 27(Q/E),43(A/S), 49(Y/F), 52(S/N), 71(F/S), 87(Y/F), and 91(S/Y).

A 10⁹ library sampled mutants with 4 or fewer mutated residues at least1 time.

The majority of the library with mutants carrying 4 to 6 residues wasmutated by doping.

-   -   rHC library: recombine outputs of H1+H2 and H3 libraries.    -   rHCLC library: recombine outputs of H1+H2, H3, and LC libraries.

Both VH and VL framework germlining reduced predicted immunogenicity.The most desirable germlining mutation in the h1A11VL.1a was the S43Aand S71F

Used Codons Specified Here for Residues that were Doped:

If a proline is to be doped, the doping oligo will haveC₍₅₋₈₅₋₅₋₅₎C₍₅₋₈₅₋₅₋₅₎S codon regardless of the original codon in theantibody sequence. These codons were selected based on the followingcriteria:

-   -   1. increase non-synonymous mutation    -   2. increase coverage of more amino acids when mutated    -   3. uses high frequency codons    -   4. avoid SSS and WWW codons.

Doping order was A-C-G-T A(85-5-5-5), A(70-10-10-10) C(5-85-5-5),C(10-70-10-10) G(5-5-85-5), G(10-10-70-10) T(5-5-5-85), T(10-10-10-70)Alanine (A): GCN G₍₁₀₋₁₀₋₇₀₋₁₀₎C₍₁₀₋₇₀₋₁₀₋₁₀₎S G₍₅₋₅₋₈₅₋₅₎C₍₅₋₈₅₋₅₋₅₎SThreonine (T): ACN A₍₇₀₋₁₀₋₁₀₋₁₀₎C₍₁₀₋₇₀₋₁₀₋₁₀₎S A₍₈₅₋₅₋₅₋₅₎C₍₅₋₈₅₋₅₋₅₎SProline (P): CCN C₍₁₀₋₇₀₋₁₀₋₁₀₎C₍₁₀₋₇₀₋₁₀₋₁₀₎S C₍₅₋₈₅₋₅₋₅₎C₍₅₋₈₅₋₅₋₅₎SSerine (S): If T₍₁₀₋₁₀₋₁₀₋₇₀₎C₍₁₀₋₇₀₋₁₀₋₁₀₎S T₍₅₋₅₋₅₋₈₅₎C₍₅₋₈₅₋₅₋₅₎S TCNIf A₍₇₀₋₁₀₋₁₀₋₁₀₎G₍₁₀₋₁₀₋₇₀₋₁₀₎C₍₁₀₋₇₀₋₁₀₋₁₀₎A₍₈₅₋₅₋₅₋₅₎G₍₅₋₅₋₈₅₋₅₎C₍₅₋₈₅₋₅₋₅₎ AGY Valine (V): GTNG₍₁₀₋₁₀₋₇₀₋₁₀₎T₍₁₀₋₁₀₋₁₀₋₇₀₎S G₍₅₋₅₋₈₅₋₅₎T₍₅₋₅₋₅₋₈₅₎S Glycine (G): GGNG₍₁₀₋₁₀₋₇₀₋₁₀₎G₍₁₀₋₁₀₋₇₀₋₁₀₎S G₍₅₋₅₋₈₅₋₅₎G₍₅₋₅₋₈₅₋₅₎S Leucine (L): IfC₍₁₀₋₇₀₋₁₀₋₁₀₎T₍₁₀₋₁₀₋₁₀₋₇₀₎S C₍₅₋₈₅₋₅₋₅₎T₍₅₋₅₋₅₋₈₅₎S CTN IfT₍₁₀₋₁₀₋₁₀₋₇₀₎T₍₁₀₋₁₀₋₁₀₋₇₀₎G₍₁₀₋₁₀₋₇₀₋₁₀₎T₍₅₋₅₋₅₋₈₅₎T₍₅₋₅₋₅₋₈₅₎G₍₅₋₅₋₈₅₋₅₎ TTR Arginine (R): IfC₍₁₀₋₇₀₋₁₀₋₁₀₎G₍₁₀₋₁₀₋₇₀₋₁₀₎S C₍₅₋₈₅₋₅₋₅₎G₍₅₋₅₋₈₅₋₅₎S CGN IfA₍₇₀₋₁₀₋₁₀₋₁₀₎G₍₁₀₋₁₀₋₇₀₋₁₀₎G₍₁₀₋₁₀₋₇₀₋₁₀₎A₍₈₅₋₅₋₅₋₅₎G₍₅₋₅₋₈₅₋₅₎G₍₅₋₅₋₈₅₋₅₎ AGR Methionine (M): ATGA₍₇₀₋₁₀₋₁₀₋₁₀₎T₍₁₀₋₁₀₋₁₀₋₇₀₎G₍₁₀₋₁₀₋₇₀₋₁₀₎A₍₈₅₋₅₋₅₋₅₎T₍₅₋₅₋₅₋₈₅₎G₍₅₋₅₋₈₅₋₅₎ Tryptophan (W): TGGT₍₁₀₋₁₀₋₁₀₋₇₀₎G₍₁₀₋₁₀₋₇₀₋₁₀₎G₍₁₀₋₁₀₋₇₀₋₁₀₎T₍₅₋₅₋₅₋₈₅₎G₍₅₋₅₋₈₅₋₅₎G₍₅₋₅₋₈₅₋₅₎ Pheylalanine (F): TTYT₍₁₀₋₁₀₋₁₀₋₇₀₎T₍₁₀₋₁₀₋₁₀₋₇₀₎C₍₁₀₋₇₀₋₁₀₋₁₀₎T₍₅₋₅₋₅₋₈₅₎T₍₅₋₅₋₅₋₈₅₎C₍₅₋₈₅₋₅₋₅₎ Isoleucine (I): need two oligos 50%A₍₇₀₋₁₀₋₁₀₋₁₀₎T₍₁₀₋₁₀₋₁₀₋₇₀₎C₍₁₀₋₇₀₋₁₀₋₁₀₎A₍₈₅₋₅₋₅₋₅₎T₍₅₋₅₋₅₋₈₅₎C₍₅₋₈₅₋₅₋₅₎ ATY 50%A₍₇₀₋₁₀₋₁₀₋₁₀₎T₍₁₀₋₁₀₋₁₀₋₇₀₎A₍₇₀₋₁₀₋₁₀₋₁₀₎A₍₈₅₋₅₋₅₋₅₎T₍₅₋₅₋₅₋₈₅₎A₍₈₅₋₅₋₅₋₅₎ ATA Tyrosine (Y): TAYT₍₁₀₋₁₀₋₁₀₋₇₀₎A₍₇₀₋₁₀₋₁₀₋₁₀₎C₍₁₀₋₇₀₋₁₀₋₁₀₎T₍₅₋₅₋₅₋₈₅₎A₍₈₅₋₅₋₅₋₅₎C₍₅₋₈₅₋₅₋₅₎ Histidine (H): CAYC₍₁₀₋₇₀₋₁₀₋₁₀₎A₍₇₀₋₁₀₋₁₀₋₁₀₎C₍₁₀₋₇₀₋₁₀₋₁₀₎C₍₅₋₈₅₋₅₋₅₎A₍₈₅₋₅₋₅₋₅₎C₍₅₋₈₅₋₅₋₅₎ Glutamine (Q): CARC₍₁₀₋₇₀₋₁₀₋₁₀₎A₍₇₀₋₁₀₋₁₀₋₁₀₎G₍₁₀₋₁₀₋₇₀₋₁₀₎C₍₅₋₈₅₋₅₋₅₎A₍₈₅₋₅₋₅₋₅₎G₍₅₋₅₋₈₅₋₅₎ Asparagines (N): AAYA₍₇₀₋₁₀₋₁₀₋₁₀₎A₍₇₀₋₁₀₋₁₀₋₁₀₎C₍₁₀₋₇₀₋₁₀₋₁₀₎A₍₈₅₋₅₋₅₋₅₎A₍₈₅₋₅₋₅₋₅₎C₍₅₋₈₅₋₅₋₅₎ Lysine (K): AARA₍₇₀₋₁₀₋₁₀₋₁₀₎A₍₇₀₋₁₀₋₁₀₋₁₀₎G₍₁₀₋₁₀₋₇₀₋₁₀₎A₍₈₅₋₅₋₅₋₅₎A₍₈₅₋₅₋₅₋₅₎G₍₅₋₅₋₈₅₋₅₎ Aspartic Acid (D): GAYG₍₁₀₋₁₀₋₇₀₋₁₀₎A₍₇₀₋₁₀₋₁₀₋₁₀₎C₍₁₀₋₇₀₋₁₀₋₁₀₎G₍₅₋₅₋₈₅₋₅₎A₍₈₅₋₅₋₅₋₅₎C₍₅₋₈₅₋₅₋₅₎ Glutamic acid (E): GARG₍₁₀₋₁₀₋₇₀₋₁₀₎A₍₇₀₋₁₀₋₁₀₋₁₀₎G₍₁₀₋₁₀₋₇₀₋₁₀₎G₍₅₋₅₋₈₅₋₅₎A₍₈₅₋₅₋₅₋₅₎G₍₅₋₅₋₈₅₋₅₎ Cysteine (C): TGY always NNS

The h1A11.1 libraries were transformed into yeast cells and displayed onthe cell surface to be selected against low concentration ofbiotinylated DLL4 extracellular domain by magnetic then fluorescenceactivated cell sorting. Selection for improved on-rate or off-rate orboth were carried out, and antibody protein sequences ofaffinity-modulated hu1A11 clones (Tables 20 and 21, below) wererecovered from yeast cells for converting back to IgG format for furthercharacterization (see, summary of clones in Table 22). Table 23 liststhe amino acids observed during the affinity maturation selection inboth framework regions (FR) and CDRs.

TABLE 20  VH sequences of Affinity Matured Humanized 1A11.1 Clones SEQ ID Sequence NO: Protein region 123456789012345678901234567890 187h1A11VH.1 EVQLVESGGGLVQPGGSLRLSC VH AASGFTFSNFPMAWVRQAPGKGLEWVATISSSDGTTYYRDSVKG RFTISRDNAKNSLYLQMNSLRA EDTAVYYCARGYYNSPFAYWGQGTLVTVSS h1A11VH.1 CDR Residues NFPMA VH -H1 31-35 of SEQ ID NO: 187h1A11VH.1 CDR Residues TISSSDGTTYYRDSVKG VH -H2 50-66 of SEQ ID NO: 187h1A11VH.1 CDR Residues GYYNSPFAY VH -H3 99-107 of SEQ ID NO: 187 188h1A11.A6 EVQLVESGGGLVQPGGSLRLSC VH AASGFTFRHFPMAWVRQAPGKGLEWVATISSSDAWPSYRDSVKG RFTISRDNAKNSLYLQMNSLRA EDTAVYYCSRGYYNSPFAYWGQGTLVTVSS h1A11.A6 CDR Residues HFPMA VH -H1 31-35 of SEQ ID NO: 188h1A11.A6 CDR Residues TISSSDAWPSYRDSVKG VH -H2 50-66 of SEQ ID NO: 188h1A11.A6 CDR Residues GYYNSPFAY VH -H3 99-107 of SEQ ID NO: 188 189h1A11.A8 EVQLVESGGGLVQPGGSLRLSC VH AASGFTFGNFPMSWVRQAPGKGLEWVASISSSDSWAT1GDSVKG RFTISRDNAKNSLYLQMNSLRA EDTAVYYCSRGYYNSPFAYWGQGTLVTVSS h1A11.A8 CDR Residues NFPMS VH -H1 31-35 of SEQ ID NO: 189h1A11.A8 CDR Residues SISSSDSWATIGDSVKG VH -H2 50-66 of SEQ ID NO: 189h1A11.A8 CDR Residues GYYNSPFAY VH -H3 99-107 of SEQ ID NO: 189 190h1A11.C6 EVQLVESGGGLVQPGGSLRLSC VH AASGFTFRNFPMAWVRQAPGKGLEWVATISSSDGWPTYRDSVKG RFTISRDNAKSSLYLQMNSLRA EDTAVYYCSRGYYNSPFAYWGQGTLVTVSS h1A11.C6 CDR Residues VH -H1 31-35 of SEQ ID NO: 190 h1A11.C6CDR Residues TISSSDGWPTYRDSVKG VH -H2 50-66 of SEQ ID NO: 190 h1A11.C6CDR Residues GYYNSPFAY VH -H3 99-107 of SEQ ID NO: 190 191 h1A11.A11EVQLVESGGGLVQPGGSLRLSC VH AASGFTFRHFPMAWVRQAPGKG LEWVATISSSDDWPNYRDSVKGRFTISRDNAKSSLYLQMNSLRA EDTAVYYCSRGYYNSPFAYWGQ GTLVTVSS h1A11.A11 CDRResidues HFPMA VH -H1 31-35 of SEQ ID NO: 191 h1A11.A11 CDR ResiduesTISSSDDWPNYRDSVKG VH -H2 50-66 of SEQ ID NO: 191 h1A11.A11 CDR ResiduesGYYNSPFAY VH -H3 99-107 of SEQ ID NO: 191 192 h1A11.B5EVQLVESGGGLVQPGGSLRLSC VH AASGFTFRYFPMSWVRQAPGKG LEWVASISGSDGWASYRDSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARGYYNSPFASWGQ GTLVTVSS h1A11.B5 CDRResidues YFPMS VH -H1 31-35 of SEQ ID NO: 192 h1A11.B5 CDR ResiduesSISGSDGWASYRDSVKG VH -H2 50-66 of SEQ ID NO: 192 h1A11.B5 CDR ResiduesGYYNSPFAS VH -H3 99-107 of SEQ ID NO: 192 193 h1A11.E12EVQLVESGGGLVQPGGSLRLSC VH AASGFTFRYFPMAWVRQAPGKG LEWVATISGSDEWPNYRDSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARGYYNSPFAFWGQ GTLVTVSS H1A11.E12 CDRResidues YFPMA VH -H1 31-35 of SEQ ID NO: 193 h1A11.E12 CDR ResiduesTISGSDEWPNYRDSVKG VH -H2 50-66 of SEQ ID NO: 193 h1A11.E12 CDR ResiduesGYYNSPFAF VH -H3 99-107 of SEQ ID NO: 193 194 h1A11.G3EVQLVESGGGLVQPGGSLRLSC VH AASGFTFRYFPMSWVRQAPGKG LEWVAS1SGSDGWASYRDSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARGYYNSPFAYWGQ GTLVTVSS h1A11.G3 CDRResidues YFPMS VH -H1 31-35 of SEQ ID NO: 194 h1A11.G3 CDR ResiduesSISGSDGWASYRDSVKG VH -H2 50-66 of SEQ ID NO: 194 h1A11.G3 CDR ResiduesGYYNSPFAY VH -H3 99-107 of SEQ ID NO: 194 195 h1A11.F5EVQLVESGGGLVQPGGSLRLSC VH AASGFTFRHFPMAWVRQAPGKG LEWVATISSSDAWPSYRDSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARGYYNSPFAYWGQ GTLVTVSS h1A11.F5 CDRResidues HFPMA VH -H1 31-35 of SEQ ID NO: 195 h1A11.F5 CDR ResiduesTISSSDAWPSYRDSVKG VH -H2 50-66 of SEQ ID NO: 195 h1A11.F5 CDR ResiduesGYYNSPFAY VH -H3 99-107 of SEQ ID NO: 195 196 h1A11.H2EVQLVESGGGLVQPGGSLRLSC VH AASGFTFGNFPMSWVRQAPGKG LEWVASISSSDSWATIGDSVKGRFTISRDNAKNSLYLQMNSLRA EDTAVYYCARGYYNSPFAFWGQ GTLVTVSS h1A11.H2 CDRResidues NFPMS VH -H1 31-35 of SEQ ID NO: 196 h1A11.H2 CDR ResiduesSISSSDSWATIGDSVKG VH -H2 50-66 of SEQ ID NO: 196 h1A11.H2 CDR ResiduesGYYNSPFAF VH -H3 99-107 of SEQ ID NO: 196

TABLE 21 VL Sequences of Affinity Matured Humanized 1A11.1 Clones. SEQ Sequence ID 123456789012345 NO: Protein region 678901234567890 197h1A11VL.1 DIQMTQSPSSLSASVGDRVTITC VL RASEDIYSNLAWYQQKPGKAPKLLIYDTNNLADGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQY NNYPPTFGQGTKLEIKh1A11VL.1 CDR Residues RASEDIYSNLA VL -L1 24-34 of SEQ ID NO: 197h1A11VL.1 CDR Residues DTNNLAD VL -L2 50-56 of SEQ ID NO: 197 h1A11VL.1CDR Residues QQYNNYPPT VL -L3 89-97 of SEQ ID NO: 197 198 h1A11.A2DIQMTQSPSSLSASVGDRVTITC VL RASQDIYINLAWYQQKPGKSPKLLIFDTNDLADGVPSRFSGSGSG TDFTLTISSLQPEDFATYYCQQY DYVPPTFGQGTKLEIKh1A11.A2 CDR Residues RASQDIYINLA VL -L1 24-34 of SEQ ID NO: 198h1A11.A2 CDR Residues DTNDLAD VL -L2 50-56 of SEQ ID NO: 198 h1A11.A2CDR Residues QQYDYVPPT VL -L3 89-97 of SEQ ID NO: 198 199 h1A11.A12DIQMTQSPSSLSASVGDRVTITC VL RASQDIYYNLAWYQQKPGKSPKLLIFDTSSLADGVPSRFSGSGSGT DFTLTISSLQPEDFATYFCQQYD WYPPTFGQGTKLEIKh1A11.A12 CDR Residues RASQDIYYNLA VL -L1 24-34 of SEQ ID NO: 199h1A11.A12 CDR Residues DTSSLAD VL -L2 50-56 of SEQ ID NO: 199 h1A11.A12CDR Residues QQYDWYPPT VL -L3 89-97 of SEQ ID NO: 199 200 h1A11.A7DIQMTQSPSSLSASVGDRVTITC VL RASQDIY1NLAWYQQKPGKAPKLLIFDTSDLADGVPSRFSGSGSGT DFTLTISSLQPEDFATYYCQQYD YYPPTFGQGTKLEIKh1A11.A7 CDR Residues RASQDIYINLA VL -L1 24-34 of SEQ ID NO: 200h1A11.A7 CDR Residues DTSDLAD VL -L2 50-56 of SEQ ID NO: 200 h1A11.A7CDR Residues QQYDYYPPT VL -L3 89-97 of SEQ ID NO: 200 201 h1A11.B4DIQMTQSPSSLSASVGDRVTITC VL RASQDIYYNLAWYQQKPGKAPKLLIFDTNILADGVPSRFSGSGSGT DFTLTISSLQPEDFATYFCQQYD YVPPTFGQGTKLEIKh1A11.B4 CDR Residues RASQDIYYNLA VL -L1 24-34 of SEQ ID NO: 201h1A11.B4 CDR Residues DTNILAD VL -L2 50-56 of SEQ ID NO: 201 h1A11.B4CDR Residues QQYDYVPPT VL -L3 89-97 of SEQ ID NO: 201 202 h1A11.B5DIQMTQSPSSLSASVGDRVTITC VL RASQDIWNNLAWYQQKPGKSP KLLIFDTSYLADGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQ YDWYPPTFGQGTKLEIK h1A11.135 CDR ResiduesRASQDIWNNLA VL -L1 24-34 of SEQ ID NO: 202 h1A11.B5 CDR Residues DTSYLADVL -L2 50-56 of SEQ ID NO: 202 h1A11.B5 CDR Residues QQYDWYPPT VL -L389-97 of SEQ ID NO: 202 203 h1A11.E12 DIQMTQSPSSLSASVGDRVTITC VLRASQEIYRNLAWYQQKPGKSPK LLIFDTSVLADGVPSRFSGSGSGT DSTLTISSLQPEDFATYYCQQYTYYPPTFGQGTKLEIK h1A11.E12 CDR Residues RASQEIYRNLA VL -L1 24-34 ofSEQ ID NO: 203 h1A11.E 12 CDR Residues DTSVLAD VL -L2 50-56 of SEQ IDNO: 203 h1A11.E12 CDR Residues QQYTYYPPT VL -L3 89-97 of SEQ ID NO: 203

TABLE 22 Summary of Affinity Matured h1A11.1 Converted Clones. Clonename VH VL h1A11.A6 h1A11.A6 VH h1A11VL.1 h1A11.C6 h1A11.C6 VH h1A11VL.1h1A11.A11 h1A11.A11 VH h1A11VL.1 h1A11.A8 h1A11.A8 VH h1A11VL.1 h1A11.B4h1A11VH.1 h1A11.B4 VL h1A11.A7 h1A11VH.1 h1A11.A7 VL h1A11.A12 h1A11VH.1h1A11.A12 VL h1A11.A2 h1A11VH.1 h1A11.A2 VL h1A11.B5 h1A11.B5 VHh1A11.B5 VL h1A11.E12 h1A11.E12 VH h1A11.E12 VL h1A11.G3 h1A11.G3 VHh1A11.E12 VL h1A11.F5 h1A11.F5 VH h1A11.E12 VL h1A11.H2 h1A11.H2 VHh1A11.E12 VL

TABLE 23 Amino acids observed during affinity maturation selections ofh1A11.1 in framework regions (FRs) and each of the CDRs for VH(SEQ ID NO: 204) and VL (SEQ ID NO: 205) regions. SEQ ID NO: 204 VHEVQLVESGGGLVQPGGSLRLSCAASGFTFSNFPMAWVRQAPGKGLEWVATISSSDGTTYYRDSVNG                              RYY S              S  G  SSASIG                              NH  T              A     AFDN                              GS                       EWST                              KA                       D PA                              T                        F  D                              L                        Q                                                       CRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGYYNSPFAYWGQGTLVTVSS          SL          D                DF S SEQ ID NO: 205 VLDIQMTQSPSSLSASVGDRVTITCRASEDIYSNLAWYQQKPGKAPKLLIYDTNNLAD                         TQE WN           S     F  NS                           T EI                     D                             DT                     Q                             NR                     T                              M                     V                              G                     EGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNNYPPTFGQGTKLEIK              S               F   SDWV  P                                   TY                                   YI                                    F                                    P

The hu1A11.1 affinity matured clones (Table 22) were expressed,purified, and further characterized in vitro. Their antigen bindingaffinities were determined by Biacore technology as described in Example1.1, and are shown in Tables 24 and 25 (below). Their activities ofbinding to cell-bound DLL4 and inhibiting cell-bound DLL4-induced Notchactivation were further examined using methods described in Examples 1.3and 1.6, and are summarized in Table 26 (below).

TABLE 24 Biacore Kinetics of Affinity-Matured Humanized 1A11.1Antibodies Binding to Human and Cynomolgus Monkey DLL4. Kinetics onBiacore Human DLL4 Cyno DLL4 k_(a) k_(d) K_(D) k_(a) k_(d) K_(D) Clone(M⁻¹s⁻¹) (s⁻¹) (nM) (M⁻¹s⁻¹) (s⁻¹) (nM) h1A11.A6 1.82 × 10⁺⁵ 6.62 × 10⁻⁶3.6 × 10⁻¹¹ 1.71 × 10⁺⁵ 2.10 × 10⁻⁵ 1.2 × 10⁻¹⁰ h1A11.C6 1.78 × 10⁺⁵7.18 × 10⁻⁶ 4.0 × 10⁻¹¹ 1.71 × 10⁺⁵ 3.12 × 10⁻⁵ 1.8 × 10⁻¹⁰ h1A11-G31.09 × 10⁺⁵ 9.39 × 10⁻⁶ 8.7 × 10⁻¹¹ 9.90 × 10⁺⁴ 1.73 × 10⁻⁵ 1.7 × 10⁻¹⁰h1A11-F5 1.31 × 10⁺⁵ 9.82 × 10⁻⁶ 7.5 × 10⁻¹¹ 1.18 × 10⁺⁵ 2.00 × 10⁻⁵ 1.7× 10⁻¹⁰ h1A11.A8 1.74 × 10⁺⁵ 1.08 × 10⁻⁵ 6.2 × 10⁻¹¹ 1.60 × 10⁺⁵ 2.40 ×10⁻⁵ 1.5 × 10⁻¹⁰ h1A11.A11 1.83 × 10⁺⁵ 2.66 × 10⁻⁵ 1.5 × 10⁻¹⁰ 1.70 ×10⁺⁵ 3.35 × 10⁻⁵ 2.0 × 10⁻¹⁰ h1A11-E12 1.49 × 10⁺⁵ 3.26 × 10⁻⁵ 2.2 ×10⁻¹⁰ 1.37 × 10⁺⁵ 3.84 × 10⁻⁵ 2.8 × 10⁻¹⁰ h1A11-H2 1.43 × 10⁺⁵ 3.85 ×10⁻⁵ 2.7 × 10⁻¹⁰ 1.31 × 10⁺⁵ 4.60 × 10⁻⁵ 3.5 × 10⁻¹⁰ h1A11-B5 1.30 ×10⁺⁵ 1.34 × 10⁻⁴ 1.0 × 10⁻⁹  1.17 × 10⁺⁵ 1.78 × 10⁻⁴ 1.5 × 10⁻⁹ h1A11.A2 1.42 × 10⁺⁵ 4.21 × 10⁻⁴ 3.0 × 10⁻⁹  1.34 × 10⁺⁵ 5.27 × 10⁻⁴ 3.9× 10⁻⁹  h1A11-B4 1.57 × 10⁺⁵ 8.23 × 10⁻⁴ 5.2 × 10⁻⁹  1.43 × 10⁺⁵ 9.90 ×10⁻⁴ 6.9 × 10⁻⁹  h1A11.A7 1.70 × 10⁺⁵ 9.73 × 10⁻⁴ 5.7 × 10⁻⁹  1.58 ×10⁺⁵ 1.24 × 10⁻³ 7.8 × 10⁻⁹  h1A11.A12 1.73 × 10⁺⁵ 1.32 × 10⁻³ 7.6 ×10⁻⁹  1.58 × 10⁺⁵ 1.62 × 10⁻³ 1.0 × 10⁻⁸  h1A11.1 1.58 × 10⁺⁵ 2.12 ×10⁻³ 1.3 × 10⁻⁸  1.44 × 10⁺⁵ 2.55 × 10⁻³ 1.8 × 10⁻⁸ 

TABLE 25 Biacore Kinetics of Affinity-Matured Humanized 1A11.1Antibodies Binding to Mouse and Rat DLL4. Kinetics on Biacore Mouse DLL4Rat DLL4 k_(a) k_(d) K_(D) k_(a) k_(d) K_(D) Clone (M⁻¹s⁻¹) (s⁻¹) (nM)(M⁻¹s⁻¹) (s⁻¹) (nM) h1A11.A6 1.98 × 10⁺⁵ 3.12 × 10⁻⁵ 1.6 × 10⁻¹⁰ 1.29 ×10⁺⁵ 7.72 × 10⁻⁴ 6.0 × 10⁻⁹ h1A11.C6 2.03 × 10⁺⁵ 2.34 × 10⁻⁵ 1.2 × 10⁻¹⁰1.69 × 10⁺⁵ 3.05 × 10⁻³ 1.8 × 10⁻⁸ h1A11-G3 1.17 × 10⁺⁵ 4.04 × 10⁻⁵ 3.5× 10⁻¹⁰ 1.18 × 10⁺⁵ 1.01 × 10⁻³ 8.6 × 10⁻⁹ h1A11-F5 1.43 × 10⁺⁵ 3.97 ×10⁻⁵ 2.8 × 10⁻¹⁰ 1.16 × 10⁺⁵ 6.79 × 10⁻⁴ 5.8 × 10⁻⁹ h1A11.A8 1.87 × 10⁺⁵3.27 × 10⁻⁵ 1.8 × 10⁻¹⁰ 1.39 × 10⁺⁵ 6.50 × 10⁻³ 4.7 × 10⁻⁸ h1A11.A111.98 × 10⁺⁵ 3.54 × 10⁻⁵ 1.8 × 10⁻¹⁰ 1.16 × 10⁺⁵ 1.02 × 10⁻³ 8.8 × 10⁻⁹h1A11-E12 1.56 × 10⁺⁵ 5.44 × 10⁻⁵ 3.5 × 10⁻¹⁰ 1.08 × 10⁺⁵ 1.75 × 10⁻⁴1.6 × 10⁻⁹ h1A11-H2 1.54 × 10⁺⁵ 5.07 × 10⁻⁵ 3.3 × 10⁻¹⁰ 1.78 × 10⁺⁵ 2.83× 10⁻³ 1.6 × 10⁻⁸ h1A11-B5 1.45 × 10⁺⁵ 1.66 × 10⁻⁴ 1.2 × 10⁻⁹  9.82 ×10⁺⁴ 3.97 × 10⁻² 4.1 × 10⁻⁷ h1A11.A2 1.81 × 10⁺⁵ 9.04 × 10⁻⁴ 5.0 × 10⁻⁹ NB NB NB h1A11-B4 4.79 × 10⁺⁵ 2.5 × 10⁻³ 5.2 × 10⁻⁹  NB NB NB h1A11.A7poor binding 1.3 × 10⁻⁸  NB NB NB h1A11.A12 poor binding 1.6 × 10⁻⁸  NBNB NB h1A11.1 1.56 × 10⁺⁵ 4.98 × 10⁻³ 3.2 × 10⁻⁸  NB NB NB NB = noobservable binding

TABLE 26 In Vitro Activities Against Cell-Bound DLL4 of Affinity-MaturedHumanized 1A11.1 Antibodies. Inhibition of Notch Direct Binding to DLL4Activation via DLL4 cells, cells, FACS (nM) Notch reporter assay (nM)Human Mouse Human Mouse DLL4 DLL4 DLL4 DLL4 h1A11.A6 2.227 0.636 0.7461.168 h1A11.C6 2.452 0.517 0.894 1.188 h1A11-G3 3.592 1.397 1.845 2.353h1A11-F5 1.171 0.460 0.484 0.649 h1A11.A8 3.160 0.744 1.331 1.247h1A11.A11 2.480 0.500 0.904 1.175 h1A11-E12 0.996 1.615 0.208 0.266h1A11-H2 1.977 0.420 0.856 0.586 h1A11.A2 2.375 0.634 3.681 0.854h1A11-B4 2.145 0.665 3.280 1.079 h1A11.A7 2.174 0.625 2.920 1.788h1A11.A12 1.768 0.568 1.662 0.832

Example 9. Molecular Identity and Physicochemical Properties of RatHybridoma Antibodies

The identity of monoclonal antibodies specific to DLL4 was determined bymass spectrometry as described below.

Mass Spectrometry Analysis of h1A11.1

The light chain molecular weight of 23,501 Daltons matched well with thetheoretical value. The heavy chain molecular weights matched well withthe theoretical values. The observed molecular weights were 50,190Daltons; 50,352 Daltons; and 50,514 Daltons, with the differencecorresponding to 162 Daltons as the result of different glycosylation.

Mass Spectrometry Analysis of h38H12.11

The light chain molecular weight of 23,408 Daltons matched well with thetheoretical value. The heavy chain molecular weights matched well withthe theoretical values. The observed molecular weights were 50,368Dalton; 50,530 Daltons; and 50,692 Daltons; with the differencecorresponding to 162 Daltons as the result of different glycosylation.

The solubilities of the antibodies were estimated by polyethylene glycol(PEG) 3000 precipitation. They were also directly determined, i.e., realsolubility, by concentrating the antibodies in a specific solutionand/or buffer with Amicon centrifugal filters and then observed for anyprecipitation at 25° C. and 5° C. Stability was inferred by nearultra-violet circular (UV-CD) and differential scanning calorimetry(DSC). Stability tofreezing and thawing and at elevated temperatures(accelerated stability) was assessed by size exclusion chromatography(SEC). The details of the techniques were described in Example 1.7, andthe results are described below.

Real Solubility Screening Results for 1A11

For a series of 1A11 clones, including hu1A11.1, hu1A11.3, hu1A11.9,hu1A11.11, and 1A11 recombinant, 2 mg of each were concentrated withAmicon centrifugal filters to above 60 mg/ml. No precipitation orcloudiness was observed at 25° C. or after storage for 1 day at 5° C.The concentrations of each were 63 mg/ml for 1A11.1, 76 mg/ml for1A11.3, 63 mg/ml for 1A11.9, 69 mg/ml for 1A11.11, and 76 mg/ml forchimeric 1A11.

Example 10. Anti-DLL4 Antibody Epitope Grouping by Biacore Technology

Epitope grouping was performed with the use of Biacore 2000, 3000, andT100 instruments. Antibodies of interest were directly immobilized onthe CMS chip surface via amino coupling. Flow cell one with similarlyimmobilized irrelevant IgG served as a reference surface. First,immobilized monoclonal antibodies (mAbs) were allowed to bindrecombinant antigen (at concentrations of at least 200 nM) for 120seconds at 50 μl/min. Then, another antibody was injected at 50 μl/mlfor 120-240 seconds to monitor its ability to bind to the antigen thatis already bound to the immobilized mAbs. The absence of additionalbinding response on the sensogram constituted overlap in the epitopes ofthe two mAbs (the one immobilized on the chip and the one introduced inliquid phase). Orientation of the assay was then switched in such a waythat the antibody that was in a liquid phase was immobilized and viceversa. The pairs of mAbs that did not allow for additional antibodybinding in both orientations of the assay were grouped as trulyoverlapping in these experiments. The resulting grouping of mAbs withoverlapping epitopes is shown in Table 27, below.

TABLE 27 Anti-DLL4 Antibody Epitope Grouping By BIAcore Technology. 1stImmobilized Injection 2nd Injection antibody huDLL4 38H12 15D6 13E4 1A1114G1 14A11 37D10 38H12 + − + + + + + + 15D6 + + − − − − − 13E4 + + − −− + − 1A11 + + − − − − − 14G1 + + + + + − − − 14A11 + + − − − − − “+”indicates binding; “−” indicates no binding

Example 11. Activities of DLL4 Antibodies in Endothelial Cell SproutingAssay In Vitro

Fibrin gel beads sprouting assay was carried out to examine the in vitroangiogenesis activity of HUVEC (passage 2-3, Lonza) as described(Nakatsu, M. N. et al. 2003 Microvasc. Res. 66, 102-112). Briefly,fibrinogen solution was reconstituted with aprotinin (4 units/ml) andthrombin (50 units/ml). Cytodex 3 beads (Amersham Pharmacia Biotech)were coated with 350-400 HUVECs per bead for over night. About 20HUVEC-coated beads were imbedded in the fibrin clot per well of a96-well tissue culture plate. Conditioned medium derived from normalhuman fibroblasts (NHLF, Lonza) at 80% confluence was plated on top ofthe gel. DLL4 antibody and control antibody KLH at 15 μg/ml were addedonto the well. At day 10 and 12, images were taken with invertedmicroscope and Nikon CCD camera. Table 28 summarizes the activities ofsome DLL4 antibodies to enhance endothelial cell sprouting in vitro.(Nakatsu et al., “Angiogenic sprouting and capillary lumen formationmodeled by human umbilical vein endothelial cells (HUVEC) in fibringels: the role of fibroblasts and Angiopoietin-1,” Microvasc. Res. 66,102-112 (2003)).

TABLE 28 Activities of DLL4 antibodies to stimulate endothelial cellsprouting. Stimulate HUVEC Tested antibody sprouting 38H12 rat mAb Yes1A11 rat mAb Yes h1A11.1 Yes 40B10 rat mAb Not observed 32C7 rat mAb Notobserved

Example 12. Rodent PK Assessment of Hybridoma-Derived Antibodies

To assess pharmacokinetics properties of anti-DLL4 antibodies,SCID-Beige mice (n=3 per antibody) were administered a singleintraperitoneal (IP) dose of antibody at either 1, 5, 10, or 30 mg/kgconcentration, depending on cross-reactivity of antibody to murine DLL4.Longitudinal serum samples (5 μl of whole blood diluted 1:50 in HBS-EP+buffer per time point) were collected from each animal over 21 days.Serum concentrations were determined using a DLL4-specific Biacoreplatform. Briefly, human DLL4 was immobilized to a sensorchip andsamples were injected over the flowcell at 5 μl per minute for 5 minuteswith the resulting binding levels measured and compared to standards.Serum concentration time profiles were used to estimate thepharmacokinetic parameters of C_(max) (peak serum concentration), CL(clearance), and t_(1/2) (antibody half-life), summarized in Table 29,below.

TABLE 29 Pharmacokinetic Parameters of Anti-DLL4 Antibodies inSCID-Beige Mice Following a Single IP Dose. Dose Cmax CL t_(1/2)Antibody (mg/kg) (μg/mL) (mL/hr/kg) (d) 38H12 rat mAb 5 30.2 0.3 20-29h1A11.1 30 163 0.44 11.3 h1A11.1 10 49.9 0.50 9.9 h1A11.1 5 11.0 1.786.3 h1A11.1 1 3.1 2.16 4.4

Example 13. DLL4 Antibody Treatment Inhibited Tumor Growth In Vivo

The effect of anti-DLL4 antibodies on tumor growth was evaluated onsubcutaneous Calu-6 xenograft tumors implanted in SCID-Beige mice.Briefly, 2×10⁶ cells were inoculated subcutaneously into the right hindflank of female SCID-Beige mice. Tumors were allowed to establish for14-18 days, at which point tumor volume was determined using electroniccaliper measurements. Tumor size was calculated using the formula:L×W²/2. Mice were allocated into treatment groups (n=10 per group) sothat each cohort of animals had equivalent mean tumor volume prior toinitiation of therapy (typically between 180 and 250 mm³). Animals werethen dosed intraperitoneally either twice a week for two weeks (total of4 doses) or weekly for four weeks (total of 4 doses) with anti-DLL4antibodies. Tumor volume was measured on average twice a week for theduration of the experiment until the mean tumor volume in each groupreached an endpoint of ≥2,000 mm³. Results are shown in Table 30, below.

TABLE 30 Efficacy of Anti-DLL4 Antibodies in the Calu-6 Human Non- SmallCell Lung Cancer Subcutaneous Xenograft Model. Dose, Route, TreatmentRegimen % T/C^(a) % ILS^(b) 1A11 rat mAb 30 mg/kg, IP, 37** 89**2X/weekX2 1A11 rat mAb 10 mg/kg, IP, 47** 39** 2X/weekX2 1A11 rat mAb 5mg/kg, IP, 43** 57** 2X/weekX2 14A11 rat mAb 10 mg/kg, IP, 37** 57**2X/weekX2 40B10 rat mAb 30 mg/kg, IP, 29** 89** 2X/weekX2 32C7 rat mAb30 mg/kg, IP, 65* 28*  2X/weekX2 14A11 chimera 10 mg/kg, IP, 32** 114** q7dX4 15D6 chimera 10 mg/kg, IP, 47** 57** q7dX4 40B10 chimera 10 mg/kg,IP, 43** 73** q7dX4 32C7 chimera 10 mg/kg, IP, 71*  18*  q7dX4 h1A11.110 mg/kg, IP, 34** 75** q7dX4 h1A11.1 5 mg/kg, IP, 31** 80** q7dX4h1A11.1 1 mg/kg, IP, 43** 36** q7dX4 h1A11.1 0.5 mg/kg, IP, 62** 25* q7dX4 ^(a)% T/C = mean tumor volume of treatment group/tumor volume oftreatment control group × 100. P values (as indicated by asterisks) werederived from Student's T test comparison of treatment group versustreatment control group. Based on day 25/26/27 measurements. ^(b)% ILS =(T − C)/C × 100, where T = median time to endpoint of treatment groupand C = median time to endpoint of treatment control group. P values (asindicated by asterisks) were derived from Kaplan Meier log-rankcomparison of treatment group versus treatment control group. Based onan endpoint of 2000 mm³. *p < 0.05; **p < 0.001

INCORPORATION BY REFERENCE

The contents of all cited references (including literature references,patents, patent applications, and websites) that may be cited throughoutthis application are hereby expressly incorporated by reference in theirentirety for any purpose, as are the references cited therein.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting of the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are therefore intended to be embracedherein.

What is claimed is:
 1. A binding protein capable of binding DLL4,comprising at least one amino acid sequence selected from the group ofamino acid sequences consisting of SEQ ID NO:157, SEQ ID NO:158, SEQ IDNO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, andSEQ ID NO:164. 2.-117. (canceled)